WO1996001120A1

WO1996001120A1 - Thymus products and applications

Info

Publication number: WO1996001120A1
Application number: PCT/GR1994/000016
Authority: WO
Inventors: Panagiotis N. Gerolymatos
Original assignee: P.N. Gerolymatos S.A.
Priority date: 1994-07-03
Filing date: 1994-07-03
Publication date: 1996-01-18
Also published as: AU7236094A

Abstract

Experimental data in the past years in immunology showed that the thymus plays an important role in the selection and development of thymocytes and in the surveillance of 'healthy' self cells of the body. Xenotransplantation (xenogeneic transplantation), surveillance and elimination of tumour cells, HIV infection and immune suppression and auto reactive cells in auto immunity are related to thymus and to the development, 'education' or maturation of thymocytes. The four projects described in this patent are novel methodological applications that are based on the central immunological role of the thymus and of the thymocytes in immune tolerance, in surveillance of cancer cells, in allogeneic tolerance and in the escape of auto reactive T cells in auto immune disease. Using different approaches targeted on the thymus activity - depending on the clinical condition - by restoring, suppressing, enhancing or by-passing this organ, therapeutical effects are aimed in clinical transplantation, cancerology, infectious diseases and auto immune disease.

Description

THYMUS PRODUCTS AND APPLICATIONS

1. XENOTRANSPLANT PROJECT

A. EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION

1. New-born human embryonic crude thymus is obtained from fresh cadavers or stili births and is used either as a crude preparation or the thymus is processed and the cells are separated and further processed.

2. The HLA haplotypes of the new-born are identified and the material is grouped according to the HLA specificity (A, C, B, DR, DQA, DQB and DP).

3. The crude thymus cells are separated by activated sorting and are placed in sealed in gas/02 flame and cooled slowly till -70 degrees centigrade. After 2-6 hours the material is transferred rapidly to a liquid N2 freezer, recorded and preserved as long as necessary. Cell lines from these cells are established and propagated in vitro.

4. Specific thymus cell lines according to HLA histocompatibility haplotypes groups are established and maintained constituting a thymus ceil bank of EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATIO .

5. The thymus cell bank maintains for each specific HLA histocompatibility group preparations for each type of thymus cell..

6. Each HLA specific EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is either crude thymus extract or a mixture of cell lines of thymocytes (at different stages of development and maturity) added to an equal number of nursing cells (epithelial, dendritic or other thymus cells).

7. The established thymus cell lines are grafted with MHC I and MHC II genes in order to produce cell lines that are marked with one or more antigenic HLA specificity. These genetically engineered thymus cell lines representing groups of

HLA specificity also constitutes an EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION.

8. The MHC I and MHC II genes are grafted using a virus based vector and a constructed replication defective retrovirus that contains complementary DNA encoding of one or more of human MHC I and MHC II antigens.

9. The EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is used to transform the miniature swine and the Pan Troglodytes (or other animal species) to chimeras of the human HLA specific histocompatible individual(s).

10. The genetically engineered to one or more MHC I and MHC II genes EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is used in an equivocal manner to transform strains of homozygotic donor animals to human HLA chimeras.

11. The EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is implanted in the thymus at birth or preferably one to three weeks (pending to the animal species) prior to the estimated delivery date by intraplacental surgery.

Alternatively the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is implanted in the bone marrow or in the spleen or is injected intravenously or in the thymus artery. The cell types that are implanted or injected belong to one or more of the established thymus cell lines and to one or more HLA specificities. 12. The EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is also used in the TUMOUR IMMUNE SUPPRESSION PROJECT, in the T CELL IMMUNE DEFICIENCY PROJECT and in the AUTO IMMUNE PROJECT. 13. The EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is introduced to the cancer, HIV immune deficient and auto immune patient in the thymus or in the

EET bone marrow or in the spleen or is injected intravenously or in the thymus artery to restore thymus functions. 14. A further indication of the EARLY HUMAN HLA SPECIFIC THYMUS CELL

PREPARATION in allogeneic transplantation. In such cases the recipient patient is treated with the HLA specific donor specificity EARLY HUMAN HLA SPECIFIC

THYMUS CELL PREPARATION prior to the transplantation and becomes a chimera to the donor. Thereafter the procedures and steps followed are the same as those described for xenotransplantation.

B. COLONY OF CHIMERICAL ANIMALS

1. At a first stage and before the establishment of the inbred strain, outbred miniature swine or Pan Troglodytes or other species is used.

2. At a second stage an homozygotic miniature swine or Pan Troglodytes strain is established and is used.

3. At a third stage by genetic engineering MHC I and MHC II genes are grafted on the animal strain's spermatozoids or oocytes prior to fertilisation establishing a new inbred strain expressing one or more human MHC I and MHC II antigens for HLA specificity. 4. Each individual animal is grouped according to the human HLA specificity to which it has been transformed chimerical to the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION.

5. The donors species are bred as laboratory animals and processed to the next two procedures (THYMUS DONOR INDIVIDUAL PREPARATION and XENOTRANSPLANTS FROM GROWN CHIMERICAL HLA - SPECIFIC ANIMALS).

6. In Table 1 are shown the combination of possibilities of the different EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION and THYMUS DONOR INDIVIDUAL PREPARATION in outbred COLONY OF CHIMERICAL ANIMALS. In Table 2 are shown the combinations for inbred homozygotic animals and in Table 3 and Table 4 for MHC I and MHC II grafted transgenic animal strains.

TABLE 1

DIFFERENT COMBINATIONS OF EARLY HUMAN HLA SPECIFIC THYMUS CELL

PREPARATION AND OF THYMUS DONOR INDIVIDUAL PREPARATION

OUTBRED ANIMAL STRAINS

@ In all cases that the outbred animals were grafted with MHC I and MHC II, the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION used is universal (for the corresponding histocompatlble MHC I and MHC II included in the graft) and there is no need that the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is specific for each HLA - human specificity Steps #1, #2, #3 and #4 are described in PROCEDURES TO BE FOLLOWED in the chapter 1.XENOTRANSPLANT PROJECT.

TABLE 2

DIFFERENT COMBINATIONS OF EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION AND OF THYMUS DONOR INDIVIDUAL PREPARATION INBRED HOMOZYGOTIC STRAINS

@ In all cases that the inbred animals were grafted with MHC I and MHC II, the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION used is universal (for the corresponding histocompatible MHC I and MHC II included in the graft) and there is no need that the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is specific for each HLA - human specificity.

* In all cases that homozygotic animals are used and were grafted with MHC I and MHC II, the THYMUS DONOR INDIVIDUAL PREPARATION is universal for all HLA histocompatible animals in relation to the histocompatibility specificity of EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION they received at birth. Therefore there is no need for specific THYMUS DONOR INDIVIDUAL PREPARATION to be prepared from each animal : the human recipient is treated prior to the xenotransplantation with a universal THYMUS DONOR INDIVIDUAL PREPARATION HLA compatible to the MHC I and MHC II antigens of the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION.

+ In all cases that homozygotic strains are used there is no need to use the same individual animals, any strain from the inbred colony can be used for xenotransplantation provided that the grafted MHC I and MHC II of the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is histocompatible to the HLA specificity of the human recipient.

TABLE 3

DIFFERENT COMBINATIONS OF EARLY HUMAN HLA SPECIFIC THYMUS CELL

PREPARATION AND OF THYMUS DONOR INDIVIDUAL PREPARATION INBRED HOMOZYGOTIC STRAINS WITH TRANSGENIC TO MHC I AND MHC II

GENES

In all situations in which inbred homozygotic transgenic MHC I and MHC II animal strains are used there is no need for either EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION or THYMUS DONOR INDIVIDUAL PREPARATION and the xenotransplantation is done directly to the human recipient from any MHC I and MHC II compatible animal strain of the colony. The transgenic animal strain grafted with MHC I and MHC II antigens that are corresponding to the human recipient's MHC I and MHC II antigens. TABLE 4

DIFFERENT COMBINATIONS OF MHC I AND MHC II GRAFTED THYMUS DONOR

INDIVIDUAL PREPARATION INBRED HOMOZYGOTIC STRAINS

In all cases that the inbred animals were grafted with MHC i and MHC II, the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION used is universal (for the corresponding histocompatible MHC I and MHC II included in the graft) and there is no need that the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is specific for each HLA • human specificity In all cases that homozygotic animals are used and were grafted with MHC I and MHC II, the THYMUS DONOR INDIVIDUAL PREPARATION is universal and there is no need for specific THYMUS DONOR INDIVIDUAL PREPARATION to be prepared from each animal : the human recipient is treated prior to the xenotransplantation with a universal THYMUS DONOR INDIVIDUAL PREPARATION compatible to the MHC I and MHC II antigens of the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION. In all cases that homozygotic strains are used there is no need to use separate individual animals,

EET any strain from the inbred colony can be used for xenotransplantation provided that the grafted MHC I and MHC II of the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is histocompatible to the HLA specificity of the human recipient.

@3 In this situation the THYMUS DONOR SPECIFIC PREPARATION is introduced to the human recipient and is recognised because of the MHC I and MHC II antigen histocompatibility as self. The reaction or rejection of the THYMUS DONOR SPECIFIC PREPARATION is minimal and the human candidate recipient becomes CHIMERIC to the MHC I and MHC II of the donor homozygotic inbred animal strain. Any xenotransplants from the corresponding animal colony MHC I and MHC II compatible can be transplanted and recognised as self both by the donor and the recipient.

@ 1 and @2 In these situations the procedure followed is the same as with inbred homozygotic strains in which crude thymus or thymus cell lines are used as EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION

C. THYMUS DONOR INDIVIDUAL PREPARATION

1. One to four weeks after the implantation or injection of the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION the animals are operated or punctured and part of their thymus extracted and used either as a crude preparation or the thymus cells are separated, sorted and processed.

2. THYMUS DONOR INDIVIDUAL PREPARATION is crude thymus, or cell lines from thymus from outbred or inbred homozygotic or transgenic donor animals.

3. The HLA specificity of the THYMUS DONOR INDIVIDUAL PREPARATION corresponds to the chimerical histocompatibility of the donor animal from which the crude thymus was extracted from. 4. The crude thymus cells are separated by activated sorting and are placed in sealed in gas/02 flame and cooled slowly till -70 degrees centigrade. After 2-6 hours the material is transferred rapidly to a liquid N2 freezer, recorded and preserved as long as necessary until the bred animal is grown up and a candidate human recipient is selected for receiving a xenotransplant from the individual animal.

5. Alternatively crude thymus is cooled, frozen and preserved without prior cell, activated sorting and used in an equivocal manner as THYMUS DONOR INDIVIDUAL PREPARATION. 6. Alternatively crude thymus extract from inbred strain of donor miniature swine or Pan Troglodytes is used for the preparation of cell lines of thymocytes, epithelial and dendritic cells and other thymus cell lines after selection and sorting. These cell lines are propagated in vitro, stored and used in an equivocal manner as THYMUS DONOR INDIVIDUAL PREPARATION. 7. Each one of these thymus cell lines corresponds to the HLA specificity of the

EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION to which the donor individual animal has become a chimera to and is maintained in the THYMUS DONOR INDIVIDUAL PREPARATION bank.

8. The preserved THYMUS DONOR INDIVIDUAL PREPARATION contains thymus cells (precursor and mature thymocytes), epithelial cells and dendritic or other thymus cells.

9. The THYMUS DONOR INDIVIDUAL PREPARATION is thawed rapidly at 37 degrees centigrade, checked for viability and implanted or injected to the candidate human recipient for xenotransplantation. The implantation or injection is in the thymus or in the bone marrow or in the spleen or intravenously or in the thymus artery.

10. The THYMUS DONOR INDIVIDUAL PREPARATION is administered to the candidate human recipient alone or if a reaction occurs concurrently with immune suppressive treatment.

10 11. Another situation is when THYMUS DONOR INDIVIDUAL PREPARATION originates from an inbred homozygotic colony that received at birth or intraplacentally EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION that was by genetic engineering grafted with one or more of MHC I and MHC II genes. In this case, the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION used is the corresponding to the HLA specificity of the MHC I and MHC II antigens included in the graft.

12. Similarly in this case the THYMUS DONOR INDIVIDUAL PREPARATION is universal and there is no need for specific THYMUS DONOR INDIVIDUAL PREPARATION to be prepared from each animal : the human recipient is treated prior to the xenotransplantation with a universal THYMUS DONOR INDIVIDUAL PREPARATION compatible to the MHC I and MHC II antigens of the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION.

13. In addition in ail such cases there is no need to use separate individual animals, any strain from the inbred colony can be used for xenotransplantation provided that the grafted MHC I and MHC II of the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is histocompatible to the HLA specificity of the human recipient. The same preparation is used for all MHC I and MHC II histocompatible candidate human recipients who in turn will receive transplants from any animal of the colony that belongs to the same homozygotic inbred chimerical strain having the same MHC I and MHC II antigens as the donor animal (Table 4).

14. Another separate situation is the use of genetic engineering to graft MHC I and MHC II to thymus cell lines of the THYMUS DONOR INDIVIDUAL PREPARATION. In this situation the THYMUS DONOR SPECIFIC PREPARATION is introduced to the human recipient and is recognised because of the MHC I and MHC II antigen histocompatibility as self. The reaction or rejection of the THYMUS DONOR SPECIFIC PREPARATION is minimal and the human candidate recipient becomes chimerical to the MHC I and MHC II of the donor homozygotic inbred animal strain. Any transplants from the corresponding animal homozygotic inbred colony MHC I

π and MHC II compatible can be transplanted and recognised as self both by the donor and the recipient. In all situations where crude thymus or thymus cell lines are used in these situations the procedure followed is the same as with inbred homozygotic strains in THYMUS DONOR SPECIFIC PREPARATION the same procedures as with homozygotic inbred strains are followed (Table 4).

15. THYMUS DONOR INDIVIDUAL PREPARATION from transgenic inbred homozygotic strains described in COLONY OF CHIMERICAL ANIMALS (paragraph 3.) constitutes a special situation. The animal strains are not introduced at birth or by transplacental surgery with EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION. These animals are chimeras by genetic engineering on their spermatozoids or oocytes to one or more MHC I and MHC II antigen HLA specificity and therefore can be given to all corresponding candidate human recipients, that are HLA histocompatible to the grafted MHC I and MHC II genes. The xenotransplantation is performed without the human recipient candidate to have to be prepared with THYMUS DONOR INDIVIDUAL PREPARATION.

Xenotransplants used are extracted from any animal of the inbred homozygotic colony without the need to use the same xenotransplant that originates from the same individual animal's THYMUS DONOR PREPARATION (Table 3).

16. The THYMUS DONOR INDIVIDUAL PREPARATION from inbred homozygotic strains is used as a universal vaccine for certain life threatening neonatal conditions in which the patient is liable to absolute need of transplantation during life. In such cases the THYMUS DONOR INDIVIDUAL PREPARATION is implanted or injected to the new-born human patient in preparation of a future transplantation.

D. XENOTRANSPLANTS FROM GROWN CHIMERICAL HLA - SPECIFIC ANIMALS 1. The donor animals are bred and grown following procedures of experimental laboratory animals, are used as a reserve of organs and tissues.

2. The patient is prepared for xenotransplantation by the administration of THYMUS DONOR INDIVIDUAL PREPARATION that corresponds to the patient's HLA specificity (except in the case of a transgenic donor).

3. The patient is transplanted one to four weeks after the administration of the THYMUS DONOR INDIVIDUAL PREPARATION

4. The xenotransplantation is performed without immune suppressive treatment. If a reaction occurs immune suppressive measures are initiated.

5. Xenotransplants from inbred homozygotic donor animal strains (that have received at birth or intraplacentally EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION containing thymus cells grafted with all MHC I and MHC II by genetic engineering) constitute a separate situation (described in Table 2 and in C. THYMUS DONOR INDIVIDUAL PREPARATION).

6. Xenotransplants from inbred homozygotic donor animal strains that by genetic engineering have been grafted at the spermatozoid or oocyte stage with MHC I and MHC II genes are transplanted to any human recipients without the need of administration of THYMUS DONOR INDIVIDUAL PREPARATION (described in Table 3 and in C. THYMUS DONOR INDIVIDUAL PREPARATION).

2. HUMAN TUMOUR IMMUNE SUPPRESSION PROJECT

A. T CELLS FROM HEALTHY INDIVIDUALS WITH HISTOCOMPATIBLE HLA HAPLOTYPES

13 1. Patient's T cells from peripheral blood, lympnatic ganglia or from the tumour site are typed for HLA specificity using allogeneic antisera or by mixed lymphocyte reaction.

2. T cell cytotoxic effect against tumoral and non tumoral cells is evaluated by the mixed lymphocyte tumour interaction test.

3. T cells of histocompatible HLA healthy volunteers are tested in the mixed lymphocyte tumour interaction test and their cytotoxic effect against tumour cells is compared to that of the putative patient's T cells.

4. Transfusion of syngeneic T cells is performed only if a higher cytotoxic effect is found in the T cells of the compatible healthy volunteer

5. Concurrently with the syngeneic T cell transfusion surgery, chemotherapy and / or radiotherapy is initiated

B. T CELL LINES OF SPECIFIC HLA - HAPLOTYPES

1. T cells from healthy volunteers are separated, sorted and typed according to their CD4+ CD8+ clone specificity, their autologus anti tumour effect and the HLA specificity.

2. Selected T cells are established as cell lines and propagated in vitro. 3. A T cell bank is established containing several or all HLA specificity cell lines. 4. T cells are preserved and used for suitable histocompatible patients showing a low putative anti tumour cytotoxic effect and a high T cell line effect.

C. GENETICALLY ENGINEERED T CELL LINES WITH GRAFTED MHC I AND MHC II GENES

1. In established and confirmed of their anti tumour cytotoxic capacity T CELL LINES OF SPECIFIC HLA - HAPLOTYPES, MHC I and MHC II genes are grafted by genetic engineering.

1 4 3. These GENETICALLY ENGINEERED T CELL LINES WITH GRAFTED MHC I AND MHC II GENES are used in MHC I and MHC II histocompatible patients with tumours under the same clinical conditions as the T CELL LINES OF SPECIFIC HLA - HAPLOTYPES.

D. EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION (as in

Xenotransplant Project)

1. New-born human embryonic crude thymus is obtained from fresh cadavers or still births and is to be used either as a crude preparation or the thymus cells are separated and processed.

3. By activated cell sorting precursor and mature thymocytes as well epithelial ("nursing") cells, and dendritic cells are isolated and separated. Cell lines from these cells are established and propagated.

4. Specific thymus cell lines according to HLA histocompatibility haplotypes groups are established and maintained constituting a thymus cell bank.

5. The thymus cell bank maintains for each specific HLA histocompatibility group preparations for each type of thymus cells.

6. Each HLA specific EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is either crude thymus extract or a mixture of cell lines of thymocytes (at different stages of development and maturity) added to an equal number of nursing cells (epithelial, dendritic or other thymus cells). 7. The established thymus cell lines are grafted with MHC I and MHC II genes in order to produce cell lines that are marked with more than one antigenic HLA specificity. These genetically engineered thymus cell lines representing groups of HLA specificity also constitute an EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION.

1 5 8. The EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is used in addition to the TUMOUR IMMUNE SUPPRESSION PROJECT, in the XENOTRANSPLANT PROJECT, in the T CELL IMMUNE DEFICIENCY PROJECT and in the AUTO IMMUNE PROJECT. 9. The EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is implanted to the patient in the thymus or in the bone marrow or in the spleen or is injected intravenously or in the thymus artery.

3. T CELL IMMUNE DEFICIENCY PROJECT

A. T CELLS FROM HEALTHY INDIVIDUALS WITH HISTOCOMPATIBLE HLA HAPLOTYPES

1. The patients are typed as to their paternal and maternal HLA haplotypes and are diagnosed if they have allogeneic T cells or other cells established in their body.

2. T cells from healthy volunteers are separated, sorted and typed according to their HLA specificity and to their cytotoxic effect against allogeneic T cells and constitute the T CELLS FROM HEALTHY INDIVIDUALS WITH HISTOCOMPATIBLE

HLA HAPLOTYPES.

3. T CELLS FROM HEALTHY INDIVIDUALS WITH HISTOCOMPATIBLE HLA HAPLOTYPES are transfused to HIV patients in which allogeneic T cells or other cells are shown to have been established in the body. 4. Selected T cells that show cytotoxic effect against the allogeneic T cells are established as cell lines and propagated in vitro to constitute the T CELL LINES OF SPECIFIC HLA HAPLOTYPES. 5. A T cell bank is established containing all or as many as possible T CELLS FROM HEALTHY INDIVIDUALS WITH HISTOCOMPATIBLE HLA HAPLOTYPES and used

16 for suitable histocompatible patients having an established allogeneic T cell population and being chimerical to one or more other HLA donor haplotypes 6. The methodology used for sorting of thymus cells, preservation of thymus cell lines and genetic engineering for grafting MHC I and MHC II genes to thymus cells is the same for all four projects.

B. MONOCLONAL ANTIBODIES AGAINST HLA HAPLOTYPES OF DONOR T CELLS

1. In patients that are chimeras to allogeneic T cells or other cells, monoclonal antibodies specific against the HLA allogeneic haplotypes are administered for a treatment period of two to six weeks. 2. The allogeneic T cells are selected and sorted and are used for the preparation of the specific syngeneic monoclonal antibodies. 3. Specific monoclonal antibodies against several T cells of specific HLA haplotypes are preserved, stocked and used for suitable histocompatible patients having an allogeneic tolerance. 4. Monitoring of the rate of elimination of the allogeneic cells from the patient will determine the length of treatment.

C. T CELL LINES OF SPECIFIC HLA HAPLOTYPES

1. T cells from healthy volunteers are separated, sorted and typed according to their CD4+ CD8+ clone specificity, their cytotoxic effect against allogeneic T cells effect and the HLA specificity. T CELLS FROM HEALTHY INDIVIDUALS WITH

HISTOCOMPATIBLE HLA HAPLOTYPES are selected, propagated and established as cell lines in vitro.

2. A T cell bank is established containing several or all HLA specificity cell lines

I 7 3. T cells are preserved and used for suitable histocompatible patients having an allogeneic tolerance.

D. GENETICALLY ENGINEERED T CELL LINES WITH GRAFTED MHC I AND MHC II GENES

1. T CELL LINES OF SPECIFIC HLA HAPLOTYPES with cytocytic effect against allogeneic T cells are selected. In established T CELL LINES OF SPECIFIC HLA - HAPLOTYPES, MHC I and MHC II genes are grafted by genetically engineering 2. These GENETICALLY ENGINEERED T CELL LINES WITH GRAFTED MHC I AND MHC II GENES are used to MHC I and MHC II histocompatible patients with HIV infections under the same clinical conditions as the T CELL LINES OF SPECIFIC HLA - HAPLOTYPES.

E. EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION (as in Xenotransplant Project)

1. New-born human embryonic crude thymus is obtained from fresh cadavers or still births and is used either as a crude preparation or the thymus cells are separated and processed.

2. The HLA haplotypes of the new-born are identified and the material is grouped according to the HLA specificity (A, C, B, DR, DQA, DQBand DP).

1 8 5. The thymus cell bank maintains for each specific HLA nistocompatibility group preparations for each type of thymus cells.

6. Each HLA specific EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is either crude thymus extract or a mixture of cell lines of thymocytes (at different stages of development and maturity) added to an equal number of nursing cells

(epithelial, dendritic or other thymus cells).

7. The established thymus cell lines are grafted with MHC I and MHC II genes in order to produce cell lines that are marked with more than one antigenic HLA specificity. These genetically engineered thymus cell lines representing groups of HLA specificity, will also constitute an EARLY HUMAN HLA SPECIFIC THYMUS

CELL PREPARATION.

8. The EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is used in addition to the T CELL IMMUNE DEFICIENCY PROJECT in the XENOTRANSPLANT PROJECT, in the HUMAN TUMOUR IMMUNE SUPPRESSION PROJECT, and in the AUTO IMMUNE PROJECT.

9. The EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is implanted to the patient in the thymus or in the bone marrow or in the spleen or injected intravenously or in the thymus artery.

4. AUTO IMMUNE PROJECT

A. T CELLS FROM SYNGENEIC HEALTHY INDIVIDUALS WITH HISTOCOMPATIBLE HLA HAPLOTYPES

1. The patients are typed as to their paternal and maternal HLA haplotypes.

2. T cells from healthy volunteers are separated, sorted and typed according to their HLA specificity and to their cytotoxic effect against auto reactive T cells and

1 9 constitute the T CELLS FROM SYNGENEIC MtALTHY INDIVIDUALS WITH HISTOCOMPATIBLE HLA HAPLOTYPES.

3. T CELLS FROM SYNGENEIC HEALTHY INDIVIDUALS WITH HISTOCOMPATIBLE HLA HAPLOTYPES are transfused to auto immune disease patients in which auto reactive ceils are confirmed.

4. A T cell bank is established containing all HLA specificity cell lines or as many as possible.

5. T cells are preserved and used for suitable histocompatible patients having auto reactive T cell population. 6. From T CELLS FROM SYNGENEIC HEALTHY INDIVIDUALS WITH

HISTOCOMPATIBLE HLA HAPLOTYPES, T CELL LINES HLA.-. HAPLOTYPE SPECIFIC are prepared.

B. T CELLS LINES HLA - HAPLOTYPES SPECIFIC

1. Selected T CELLS FROM SYNGENEIC HEALTHY INDIVIDUALS WITH HISTOCOMPATIBLE HLA HAPLOTYPES that show cytotoxic effect against auto reactive T cells are separated, sorted and typed according to their CD4+ CD8+ clone specificity and their HLA specificity are established as cell lines and are propagated in vitro.

2. These elected T cell lines constitute the T CELLS LINES HLA - HAPLOTYPES SPECIFIC.

3. A T cell bank is established containing several or all of the T CELLS LINES HLA HAPLOTYPES SPECIFIC. 4. The T CELLS LINES HLA - HAPLOTYPES SPECIFIC are preserved and used for suitable histocompatible patients.

C. MHC I AND MHC II GRAFTED T CELL LINES

20

SHEET 1. In established and confirmed of their activity against auto reactive T CELLS LINES HLA - HAPLOTYPES SPECIFIC, MHC I and MHC II genes are grafted by genetically engineering.

2. These GENETICALLY ENGINEERED T CELL LINES WITH GRAFTED MHC I AND MHC II GENES are used in MHC I and MHC II histocompatible patients with auto immune disease under the same clinical conditions as the T CELL LINES OF SPECIFIC HLA - HAPLOTYPES.

D. MONOCLONAL ANTIBODIES AGAINST HLA HAPLOTYPES OF AUTO REACTIVE T CELLS

1. In patients that have auto reactive T cells , monoclonal antibodies specific against the HLA auto reactive T cells are administered for a treatment period of two to six weeks. 2. The auto reactive T cells are selected and sorted and are used for the preparation of the specific syngeneic monoclonal antibodies.

3. Specific monoclonal antibodies against several T cells of specific HLA haplotypes are preserved, stocked and used for suitable histocompatible patients having an auto reactive T cells. 4. Monitoring of the rate of elimination of the auto reactive T cells from the patient will determine the length of treatment.

E. EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION (as in Xenotransplant Project)

2 I 2. The HLA haplotypes of the new-born are identified and the material is grouped according to the HLA specificity (A, C, B, DR, DQA, DQB and DP).

5. The thymus cell bank maintains for each specific HLA histocompatibility group preparations for each type of thymus cells. 6. Each HLA specific EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is either crude thymus extract or a mixture of cell lines of thymocytes (at different stages of development and maturity) added to an equal number of nursing cells (epithelial, dendritic or other thymus cells).

7. The established thymus cell lines are grafted with MHC I and MHC II genes in order to produce cell lines that are marked with more than one antigenic HLA specificity. These genetically engineered thymus cell lines representing groups of HLA specificity will also constitute an EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION.

8. The EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is used in addition to the AUTO IMMUNE PROJECT in the XENOTRANSPLANT PROJECT, in the TUMOUR IMMUNE SUPPRESSION PROJECT and in the T CELL IMMUNE DEFICIENCY PROJECT.

9. The EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is implanted to the patient in the thymus or in the bone marrow or in the spleen or is injected intravenously or in the thymus artery.

22 REMARKS

The methodology used for the extraction of thymus, preservation of crude thymus, sorting of thymus cells, preservation in cell banks of thymus cell lines and genetic engineering for grafting MHC I and MHC II genes to thymus cells is the same for all four projects.

23

SUBSTITUTE SHEET 1. XENOTRANSPLANT PROJECT

SCOPE

The patent describes a novel approach to achieve successful discordant xenograft transplants from miniature swine, primate Pan Troglodytes, or other animal species to human recipients. The methodology described aims to limit the complications observed up to now in transplantation surgery and to offer an abundant source of donor organs and tissues.

An adaptation of the XENOTRANSPLANTATION PROJECT allows the transplantation of organs and tissues from one human to another (allogeneic transplantation).

RESEARCH AND DEVELOPMENT PRODUCTS AND SYSTEMS

A. EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION

B. COLONY OF CHIMERICAL ANIMALS

C. THYMUS DONOR INDIVIDUAL PREPARATION

D. XENOTRANSPLANTS FROM GROWN CHIMERICAL HLA - SPECIFIC ANIMALS OBJECTIVES

The overall objective is to develop a colony (outbred or inbred homozygotic or transgenic) of donor animals that is used as an abundant reserve of organs and tissues for xenograft transplantation to humans. Specific project objectives are:

1. Use of HLA haplotypes specific thymus extracts from human new-borns to transform outbred or inbred animals to human specific HLA chimeras

2. Prepare from these HLA haplotypes specific thymus cell lines that can replace the crude extracts in the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION.

3. Prepare from these cell lines by genetic engineering cell lines with grafted MHC I and MHC II genes. These biotechnology cell lines are including and correspond to one or more human HLA haplotypes and replace the crude thymus extracts and the thymus cell lines in the HUMAN HLA SPECIFIC THYMUS CELL PREPARATION

4. Prepare from the animal donor chimeras a THYMUS DONOR INDIVIDUAL PREPARATION that transforms the human recipient to a chimera to the specific donor animal from which the transplant will be taken from.

5. Develop XENOTRANSPLANTS FROM GROWN CHIMERICAL HLA - SPECIFIC ANIMALS that are recognised as self by the human recipient and that recognise as self the cells of the human recipient.

BACKGROUND

2 5 The number of patients that are candidates for transplantation is increasing constantly world-wide due to improvements in medical science and procedures and changing epidemiology patterns in nosology and epidemiology. The demand for donor organs has reached a point that it has become a public concern and the World Health Organisation is closely monitoring the problem alarmed by reports of criminal marketing of donor organs.

Allogeneic transplants from human donors and xenografts are liable to rejection due to an immunologic response that has to be eliminated in order to achieve tolerance of a transplant. Immunologic response can be eliminated by finding matching donor-recipient combinations of identical histocompatibility antigens as is the case of identical twins. Another way to overcome the rejection response is by immune suppression. A third way to overcome the response is by creating a specific immunologic tolerance. The method described in this patent aims in creating positive and negative selection tolerance and generate a new T cell chimerical repertoire in the human recipient that will be transplanted with a xenograft from a discordant donor (miniature swine, Pan Troglodytes or other animals).

Inbred Pan Troglodytes is a difficult species to be used - at least at the beginning - as a laboratory animal, on the contrary outbred or inbred miniature swine have clear-cut and well described advantages as to their size, physiology, anatomy and breeding. The method described is equally applicable to both Pan Troglodytes and to miniature swine or outbred animals that are used as donors to human recipients.

PATENT IS BASED ON THE FOLLOWING METHODOLOGIES

1. Development of the inbred animal donor colonies.

2. Development of technology to obtain rapid testing for lymphocyte HLA haplotypes histocompatibility profile of subjects.

26 3. Development of technology to reproduce the thymus differentiation, maturation "nursing" and "education" of T cells extra corpus.

4. Development of technology to prepare from crude thymus extracts of cell lines of thymus epithelial cells, dendritic cells, precursor and mature thymocytes or other thymus cells.

5. Development of technology to obtain and preserve crude tissue and/or cell lines of thymus cells.

6. Development of technology to implant, micro - inject or install in the thymus (intra thymically) or in the bone marrow or in the spleen or in the blood by intravenous injection or by injection in the thymus artery of crude thymus or thymus cell lines or genetically engineered thymus cells and neutralise immediate rejection.

7. Development of technology to extract or implant thymus intraplacentally

8. Development by genetic engineering of methodology to graft MHC I and MHC II genes by constructing replication defective virus containing complementary DNA encoding human MHC I and MHC II antigens and induce chimerism to thymus cell lines and spermatozoids or oocytes of homozygotic animal strains.

INNOVATIONS AND/OR INVENTIONS DESCRIBED IN THE PATENT

The following innovations and/or inventions of products or systems or methodologies are described in this patent :

A. EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION

Crude thymus is obtained from new-born or still birth human cadavers as early as possible and the thymus extract is either preserved to be used as crude or it is processed and thymus cells are prepared.

2 7 Epithelial, dendritic, precursor and mature thymocyte and other cell lines from the crude thymus are contained in the crude extracts and are separated, sorted, established and propagated in vitro in order to obtain specific HLA - haplotypes cell lines.

The thymus tissue extracted from the new-bom is used: a) crude b) or is processed for the preparation of cell lines c) or the cell lines are grafted by genetic engineering with MHC I and MHC II genes .

All three thymus preparations are used in an equivocal manner as alternative constituents of the EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION. Each preparation is HLA - haplotypes' specific.

The EARLY HUMAN HLA - SPECIFIC THYMOCYTE PREPARATION is implanted in the thymus or in the bone marrow or in the spleen or is injected intravenously or in the thymus artery of inbred homozygotic or outbred new-bom donor animals or is implanted or injected by transplacental surgery to the embryo.

The EARLY HUMAN HLA - SPECIFIC THYMOCYTE PREPARATION is also used in the HUMAN IMMUNE TUMOUR SUPPRESSION PROJECT, in the IMMUNE DEFICIENCY PROJECT and in the AUTO IMMUNE PROJECT. The same preparation is also used for the preparation of patients to receive allogeneic transplants. In such cases the human recipient is prepared with EARLY HUMAN HLA - SPECIFIC THYMOCYTE PREPARATION histocompatible to the donor human prior to the transplantation and thereafter the same procedure is followed as in xenotransplantations.

B. COLONY OF CHIMERICAL DONOR ANIMALS

2 8 UTE SHEET The animals are brought up as an inbred homozygotic colony or as outbred animals

Miniature swine or Pan Troglodytes or other animals are transformed to chimeras to human HLA specificities and used as donors of organs and tissues to histocompatible human recipient patients.

The donor animals receive during the late gestation period by transplacental surgery or as new-borns, human thymus crude cells or ceil lines or genetically engineered thymus cells (EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION).

The successful implantation or injection of such material transforms the animals to chimeras to one specific or groups of human HLA - haplotypes in concordance to the constituents of the EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION that they have received in their early life.

A few days or weeks after the implant or injection to the animal of the EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION, part of their thymus is extracted for the preparation of the THYMUS DONOR INDIVIDUAL PREPARATION.

I

C. THYMUS DONOR INDIVIDUAL PREPARATION Inbred homozygotic or outbred animals are used that have been rendered chimerical to HLA - haplotypes as new-boms or prenatal. The animals are bred under laboratory conditions and after a period of one to two weeks are operated and part of the thymus extracted.

Crude thymus, thymus cell lines or thymus cell lines are processed and preserved in a THYMUS DONOR INDIVIDUAL PREPARATION bank. From the cell lines MHC I and MHC II genes are grafted and cell lines are prepared that have HLA specificity antigen markers on their surface. The preparations maintained in the bank are identifiable as

29

STITUTE SHEET to the HLA specificity to which they correspond and as to the animal identity from which extracted.

The use of THYMUS DONOR SPECIFIC PREPARATION prepared from homozygotic inbred animals having received at birth MHC I and MHC II grafted EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION and also having been grafted with corresponding MHC I and MHC II genes constitute a separate situation. In this case the candidate recipient patient will receive syngeneic THYMUS DONOR SPECIFIC PREPARATION and will recognise it as self without reaction or need of immune suppressive treatment. The THYMUS DONOR SPECIFIC PREPARATION in a similar manner recognises as self the human recipient.

In all other cases where outbred or inbred colony animals have received crude thymus or cell lines of HLA specific EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION, the introduction of the THYMUS DONOR SPECIFIC PREPARATION to the recipient patient might require immune suppressive treatment.

Another special situation is when transgenic to MHC I and MHC II inbred homozygotic animals are used. In this situation there is no need to prepare or administer THYMUS DONOR SPECIFIC PREPARATION.

The candidate patient recipient is treated with the specific THYMUS DONOR INDIVIDUAL PREPARATION. The THYMUS DONOR INDIVIDUAL PREPARATION is implanted in the thymus or bone marrow or spleen or is injected intravenously or in the thymus artery. When successfully tolerated by the human recipient candidate prior to the xenotransplantation the xenotransplantation of the histocompatible donor organ or tissues follows.

30 The THYMUS DONOR INDIVIDUAL PREPARATION is used in conjunction with plasmophoresis, monoclonal anti-human T cells, irradiation or pharmaceutical immune suppression, if required, pending on the human recipient's condition, immune status and concomitant diseases.

D. XENOTRANSPLANTS FROM GROWN CHIMERICAL HLA - SPECIFIC ANIMALS Outbred or inbred homozygotic or transgenic animals that have been rendered chimerical to HLA - haplotypes and have been grown in laboratory conditions are used.

Organ or tissue xenotransplants are extracted and transplanted to the human recipient patient that is HLA - haplotype compatible to the specific donor chimerical animal (more precisely to the HLA - haplotypes to which the individual animal had been rendered chimerical at birth).

Each animal is identifiable and corresponds to the EARLY HUMAN HLA DONOR THYMUS PREPARATION to which it was induced at birth and to the THYMUS DONOR INDIVIDUAL PREPARATION that was extracted from the animal in early life.

In the case of transgenic strains each animal is identifiable by the MHC I and MHC II genes to which it was rendered transgenic to.

In the case of homozygotic inbred strains rendered chimerical by introduction of

EARLY HUMAN HLA DONOR THYMUS PREPARATION MHC I and MHC II grafted cell lines, the animals are identifiable by the corresponding MHC I and MHC II antigen markers.

31 Five special situations are described where the THYMUS DONOR INDIVIDUAL PREPARATION has a different use :

1. In the case of COLONY OF CHIMERICAL ANIMALS that are inbred homozygotic strains and are transgenic chimeras by spermatozoid or oocyte genetic engineering to one or more MHC I and MHC II haplotypes there is no need to prepare the human recipient prior to xenotransplantation with THYMUS DONOR INDIVIDUAL PREPARATION. Xenotransplants to be used are extracted from any animal of the inbred homozygotic colony without the need to use the same xenotransplant that originates from the same individual animal's THYMUS DONOR PREPARATION.

2. In the case of THYMUS DONOR INDIVIDUAL PREPARATION that originates from an inbred homozygotic colony that received at birth or intraplacentally EARLY

HUMAN HLA SPECIFIC THYMUS CELL PREPARATION that was by genetic engineering grafted with one or more of MHC I and MHC II genes . In this case the THYMUS DONOR INDIVIDUAL PREPARATION is universal and there is no need for specific THYMUS DONOR INDIVIDUAL PREPARATION to be prepared from each animal : the human recipient is treated prior to the xenotransplantation with a universal THYMUS DONOR INDIVIDUAL PREPARATION compatible to the MHC I and MHC II antigens of the EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION. Furthermore in this situation there is no need to use separate individual animals, any strain from the inbred colony can be used for xenotransplantation provided that the grafted MHC I and MHC II of the EARLY

HUMAN HLA SPECIFIC THYMUS CELL PREPARATION is histocompatible to the HLA specificity of the human recipient. The same preparation is used for all MHC I and MHC II histocompatible candidate human recipients who in turn will receive transplants from any animal of the colony that belongs to the same homozygotic

3 2 inbred strain chimerical having the same MHC I and MHC II antigens as the donor animal.

3. In this situation the THYMUS DONOR SPECIFIC PREPARATION is introduced to the human recipient and is recognised because of the MHC I and MHC II antigen histocompatibility as self. The reaction or rejection of the THYMUS DONOR SPECIFIC PREPARATION is minimal not requiring immune suppressive measures and the human candidate recipient becomes chimerical to the MHC I and MHC II of the donor homozygotic inbred animal strain. Any transplants from the corresponding animal homozygotic inbred colony MHC I and MHC II compatible can be transplanted and recognised as self both by the donor and the recipient (Table 4)).

4. In all situations where crude thymus or thymus cell lines are used in these situations the procedure followed is the same as with inbred homozygotic strains in THYMUS DONOR SPECIFIC PREPARATION the same procedures as with homozygotic inbred strains are followed (Table 1, Table 2, and Table 3.

5. This short cut procedure is also followed in case of difficulties in achieving tolerance of the THYMUS DONOR INDIVIDUAL PREPARATION by the human recipient candidate.

PROCEDURESANDSTEPSTOBEFOLLOWED

The procedures and steps to be followed are

STEP 1

33 INTRODUCTION OF EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION TO PERINATAL DONOR ANIMAL

1. The EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION is extracted from new-born human cadavers or from still births.

2. The crude thymus is typed to identify the HLA haplotypes specificity and is either used crude or thymus cell lines are prepared and propagated in vitro as HLA specific cell lines.

3. From these cell lines by genetic engineering MHC I and MHC II genes are grafted to the different thymus cell lines.

4. The EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION can either be crude thymus or thymus cell lines or genetically engineered cell lines

5. The EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION is preserved and maintained in a bank for future introduction to animal strains. 6. In genetically engineered thymus cell lines grafted with MHC I and MHC II genes the number of EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION will be either one or more preparation or groups of HLA specificities according to the number of genes that have been successfully grafted to the thymus cells.

7. The EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION bank maintains and has on stock as many as possible HLA haplotypes of thymus or thymus cell lines.

8. The EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION is introduced or implanted to the donor animal in the thymus ally (or in the bone marrow or in the spleen or is injected intravenously or in the thymus artery). 9. The introduction to the new-bom or embryo animal of the new-born human thymus material is performed by transplacental surgery or immediately after birth 10. The future xenotransplant donor animals becomes a chimera and is tolerant to human HLA - haplotypes specific.

34 11. If the introduction is performed early during the thymus development then the donor animals will educate and produce thymocytes that are positively and negatively selected to both animal strains individual haplotype as well as the human HLA -specific haplotype from which it received thymus material. 12. The introduction consists of a graft of crude thymus or of cell lines of the EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION.

13. The introduction or implantation is performed by a multisite micro- injection, or implantation.

14. EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION is specific HLA-A, B, C, E, F, G, H for class I and HLA-D for class II MHC specific. This material contains precursor and mature thymocytes, epithelial nursing cells, dendritic, or other thymus cells.

15. The chimerical donor animals are used as a reservoir of organs and tissues that will be used in transplantation to human recipients. 16. The EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION due to the early stage of life of the donor animal is established in the donor animal and will process donor thymocytes and T cell by positive and negative selection. The donor animal becomes chimerical and its T cells recognise as self both the original donor animal cells as well as the cells of the human HLA - haplotype to which it was exposed.

17. The restriction repertoire of the donor animal's T cells is determined by the chimerical environment in which they have matured which will represent a positive and negative selection compatible to the human HLA - haplotypes and of the inbred animal donor strain's histocompatibility specificity. 18. By using MHC I and MHC II grafted genes on the human new-bom thymus cell lines the methodology is simplifies and there will be less need for use of cadavers and still births.

35 STEP 2

EXTRACTION FROM EACH INDIVIDUAL CHIMERIC ANIMAL PART OF THYMUS AND

PREPARATION OF THYMUS DONOR INDIVIDUAL PREPARATION

1. The animals are under control for a period of one to four weeks before part of their thymus is extracted and THYMUS ANIMAL DONOR INDIVIDUAL MATERIAL from each individual animal is processed and preserved till the time when the animals will be sacrificed and serve as donors for candidate human recipients. 2. From the extracted crude thymus the THYMUS DONOR INDIVIDUAL PREPARATION is processed.

3. From the same crude extracted donor animal thymus, cell lines of thymus epithelial nursing cells, dendritic cells precursor and mature thymocytes and other thymus cells are prepared, established and propagated in vitro. Cell mixtures of these cell lines are used alternatively as THYMUS DONOR

INDIVIDUAL PREPARATION instead of crude thymus.

4. From the thymus cell lines using genetic engineering, cell lines of thymus cells are grafted with one or more MHC I and MHC II genes corresponding to one or more HLA - haplotypes specificities. 5. Each THYMUS DONOR INDIVIDUAL MATERIAL corresponds to the specific human HLA - specificity or specificities to which the animal was transformed chimerical to at birth. 6. Banks of THYMUS DONOR INDIVIDUAL PREPARATION are organised that maintain the following: a. FROM OUTBRED COLONIES Crude thymus or established cell lines of thymus cells from donor animal that is individual for each animal and corresponds to the HLA specificity of the EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION. This THYMUS DONOR INDIVIDUAL PREPARATION is preserved and is introduced to a human recipient compatible to the same HLA

36 specificity. The human recipient is transplanted later with a transplant originating from the same individual animal b. FROM INBRED HOMOZYGOTIC COLONIES Genetically grafted cell lines with MHC 1 and MHC II genes from these animal strains are used as THYMUS DONOR INDIVIDUAL PREPARATION for the individual or groups of

HLA haplotypes to which the cells are compatible to. 7. Alternatively in the situation that the inbred colony is homozygotic and transgenic grafted with MHC I and MHC II genes as a spermatozoid or oocyte, there is no need for the use of THYMUS DONOR INDIVIDUAL PREPARATION. 8. In the situation that inbred homozygotic strains have received EARLY HUMAN

HLA SPECIFIC THYMUS PREPARATION with genetically engineered thymus cells with grafted MHC I and MHC II genes compatible to the MHC I and MHC II markers of the recipient patient. The candidate recipient patient will receive the histocompatible xenotransplant THYMUS DONOR INDIVIDUAL PREPARATION (grafted with MHC I and MHC II genes) and will recognise it as self without reaction or need of immune suppressive treatment.

STEP 3 TREATMENT OF HUMAN RECIPIENT WITH THYMUS DONOR INDIVIDUAL PREPARATION PRIOR TO XENOTRANSPLANTATION

1. THYMUS DONOR INDIVIDUAL PREPARATION, is introduced to the human recipient patient who is a candidate for xenotransplantation prior to the transplant intervention.

2. The tolerability is higher in the case that compatible MHC I and MHC II grafted cells are used in both the EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION and the THYMUS DONOR INDIVIDUAL PREPARATION.

37 3. In other cases the treatment with THYMUS DONOR INDIVIDUAL PREPARATION requires immune suppressive measures concurrently.

4. The human recipient by receiving prior to the xenotransplantation the THYMUS.

5. DONOR INDIVIDUAL PREPARATION is rendered chimerical to the individual donor animal. The human recipient's chimerical thymus educates and nurses accordingly the thymocytes that will be appropriately positively and negatively selected in order to recognise as self both the "human" self haplotype as well as the individual donor animal's haplotypes.

STEP 4

XENOTRANSPLANTS FROM GROWN CHIMERICAL HLA - SPECIFIC ANIMALS

1. The organs and tissues of each chimerical donor animal are transplanted to human recipient candidates.

2. The chimerical animal used for xenotransplantation is the same one from which the THYMUS DONOR INDIVIDUAL MATERIAL was prepared from, and implanted (or injected) to the human recipient candidate (or alternatively thymus cell lines or genetically engineered thymus cell lines).

3. When the xenotransplantation is performed the histocompatibility sequence of events corresponds to one or more of the following situations : a. EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION and candidate recipient patient share the same HLA specificity as to the marker MHC I and MHC II antigens. b. New-born donor animal, THYMUS DONOR INDIVIDUAL PREPARATION and Xenotransplants belong to the same individual animal.

38 c. New-born donor animal, THYMUS DONOR INDIVIDUAL PREPARATION and Xenotransplants do not belong to the same individual animal however are originating from homozygotic inbred strains. d. New-born donor animal and Xenotransplant are transgenic homozygotic inbred strains grafted possessing MHC I and MHC II antigen markers identical to the human recipient's corresponding MHC I and MHC II markers. e. EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION, MHC I and MHC II grafted cell line THYMUS DONOR INDIVIDUAL PREPARATION and human recipient patient possess identical MHC I and MHC II marker antigens. 4. The xenotransplant being a chimera of the same specific HLA - haplotype as the human recipient candidate recognises the human recipient's cells as self and is tolerated. 5. In the special situations of homozygotic inbred transgenic animal strains with grafted MHC I and MHC II genes the introduction to the human recipient of

THYMUS DONOR INDIVIDUAL PREPARATION is omitted. The MHC I and MHC II markers of the transgenic strain should include the MHC I and MHC II markers of the human recipient. 6. Step #2 and step #3 will also be omitted in the case of difficulties in achieving tolerance of the THYMUS DONOR INDIVIDUAL PREPARATION by the human recipient candidate. 7. In all these situations the xenotransplant will be directly transplanted to the human recipient.

39 2. HUMAN TUMOUR IMMUNE SUPPRESSION PROJECT

SCOPE

The patent describes a novel approach in the therapy of human tumours. In patients with tumours where there is a defect in the immune surveillance system a specific HLA - haplotypes preparation of mature T cells will be transfused in order to restore the immune surveillance system and by an enhanced cytotoxic effect destroy the tumour c cells.

RESEARCH AND DEVELOPMENT PRODUCTS AND SYSTEMS

A. T CELLS FROM HEALTHY INDIVIDUALS WITH HISTOCOMPATIBLE HLA - HAPLOTYPES

B. T CELL LINES OF SPECIFIC HLA - HAPLOTYPES

C. GENETICALLY ENGINEERED T CELL LINES WITH GRAFTED MHC I AND MHC II GENES

D. EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION (as in Xenotransplant Project)

40 PATENT IS BASED ON THE FOLLOWING METHODOLOGIES TO BE DEVELOPED:

The methodology to be developed is the same as described in the Xenotransplant Project.

INNOVATIONS AND/OR INVENTIONS DESCRIBED IN THE PATENT

The following innovations and/or inventions of products or systems or methodologies are described in this patent:

A. T CELLS FROM HEALTHY INDIVIDUALS WITH HISTOCOMPATIBLE HLA - HAPLOTYPES

The patient's histocompatibility profile is determined and the paternal and maternal HLA - haplotypes is identified by serology or mixed lymphocyte culture. This will allow to administer to the patient the suitable histocompatible healthy T cells or cell line or genetically engineered HLA - haplotypes specific anti tumour T cells.

The cytotoxic anti tumour effect of the patient's T cells and T cells of histocompatible healthy subjects or cell line T cells is compared.

Tumour cells are tested in a mixed cell culture system with histocompatible lymphocytes from normal donors and their anti-neoplastic cell cytotoxicity is determined in comparison to that of the patient's putative lymphocytes. This will allow to test the natural cytotoxic effect of the "healthy" lymphocytes that should be significantly more potent than the putative patient's T cells in order to justify the therapeutic T cell transfusion which is described in this patent.

4 I Lymphocytes from histocompatible individuals of the same HLA - haplotype is transfused to patients with human tumours to revitalise the patient's lacking immune surveillance suppressive capacity against the tumour cells.

B. T CELL LINES OF SPECIFIC HLA - HAPLOTYPES

Cell lines of mature T cells of specific HLA - haplotypes will be established and propagated in vitro allowing an abundant resource of suitable T cells that can be propagated in vitro. The selection of the T cells clones for in vitro propagation will be based on the anti tumour cytotoxic effect profile as determined by the mixed lymphocyte tumour inactivation. A T cell bank will be established in which in vitro propagated and preserved T cells of different HLA specificities will be available for transfusion to suitable patients.

The T cells are transfused in monotherapy or in combination with surgery, chemotherapy and or radiotherapy.

C. GENETICALLY ENGINEERED T CELL LINES WITH GRAFTED MHC I AND MHC II GENES

Using genetic engineering, MHC I and MHC II genes will be grafted on the propagated T cells to render them universal donors or at least compatible with large groups of HLA - haplotypes. Such a genetic manipulation will allow to limit the number of T cell lines and obtain T cells suitable for large groups of HLA - haplotypes.

D. EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION (as in Xenotransplant Project)

4 2 The preparation will be the same as the one in the Xenotransplant Project. Cell lines or genetically engineered cells are developed in addition to crude thymus extracts. Implantation is made in the thymus or in the bone marrow or in the spleen or is injected intravenously or in the thymus artery. The aim is to restore and re-educate the putative Tcells of the patient in the immune surveillance of the self cells of the body and in the destruction of neoplastic cells.

PROCEDURESANDSTEPSTOBEFOLLOWED

The theoretical approach of using the products, systems and cell preparations described is to overcome a deficiency in the immune surveillance mechanisms of the patient. By determining the patent's HLA - haplotypes candidate donor lymphocytes can be selected.

The establishment of the anti-tumour cytotoxic effect of the donor lymphocytes will determine the usefulness of the proposed therapeutic approach. Development of cell lines of T cells of different HLA - haplotypes will enable to obtain an abundant source of cells for massive treatments. HLA histocompatible lymphocyte treatment can be combined with other chemotherapeutic, radiotherapeutic or surgical approaches. Re-education of the patient's thymocytes in order to restore in the long term the immune surveillance system. Another dimension of the same project and using the same products and systems is its expansion to treat infections due to virus, bacteria, fungi and parasites.

In all infectious diseases where it can be shown that a deficiency in T cell function occurs directly or indirectly related to the development and maturation of thymocytes in the thymus then the products and systems described in this patent on human tumour immune suppression are applied in conjunction with anti-infectives or alone.

43 3. T CELL IMMUNE DEFICIENCY PROJECT

SCOPE

The patent describes a set of systems, tests and cell products and preparations that will allow to diagnose immune deficiencies and treat HIV infection based on the concept that HIV infection is secondary to an allogeneic immune condition.

The project aims in providing means to allow the patient to restore and re-establish the immunologic status of self and non-self recognition of the immune deficient state, and by an indirect way attack the HIV infection through the antigen presenting cells. The project is based on the assumption that immune deficiency is a consequence of the introduction of allogeneic T cells in the individual that is rendered a chimera.

RESEARCH AND DEVELOPMENT PRODUCTS AND SYSTEMS

A. T CELLS FROM HEALTHY INDIVIDUALS WITH HISTOCOMPATIBLE HLA HAPLOTYPES

B. MONOCLONAL ANTIBODIES AGAINST HLA HAPLOTYPES OF DONOR T CELLS

C. T CELL LINES OF SPECIFIC HLA HAPLOTYPES

44

BACKGROUND

Introduction of fresh living cells - of the innate or of the adaptive immune system - from another genetically different member of the same species can happen in: Polytransfused patients receiving fresh blood Homosexuals or heterosexuals during rectal intercourse Drug addicts by using syringes containing blood from other individuals

Infected new-boms by tranplacental passage Mosquito biting

FIRST common factor in all these groups of individuals is the introduction of allogeneic white blood cells and other donor cells in their circulation at optimal temperature and rapidly after the vehicle The vehicle can be :

Fresh blood transfusion

Remaining of blood in syringes and other material used on a multiple basis

Spermatic cells - including white blood cells -that penetrate through the dilated rectal tissues

Remaining of blood from the sucking apparatus of mosquitoes

4 5 SECOND common factor is that these groups belong to the high risk groups of AIDS patients suffering from the disease

PATENT IS BASED ON THE FOLLOWING METHODOLOGIES TO BE DEVELOPED:

INNOVATIONS AND/OR INVENTIONS DESCRIBED IN THE PATENT

A. T CELLS FROM HEALTHY INDIVIDUALS WITH HISTOCOMPATIBLE HLA HAPLOTYPES The HLA profile of each patient is evaluated by:

a) Determining the paternal and maternal initial haplotypes of the subject and to determine if the subject has become a chimera after introduction and tolerance of thymocytes from other allogeneic individuals.

b) Determining the allogeneic haplotypes installed and tolerated in the individual.

46 The allogeneic thymocytes could have been introduced by transfusion, introduction of sperm in the circulation, accidental injection, trauma or bite. In the case that the allogeneic cells have not been destroyed as non - self cells and have managed to become tolerant, then the individual is a chimera to the donor's thymocyte material to which he or she has been exposed.

T cells from healthy individuals with compatible HLA - haplotypes are transfused in order to potentate the self thymocytes and enhance the infectious control. These T cells by being HLA compatible to the original putative T cells of the patient will supply the eliminated or reduced patient's T cells and destroy the allogeneic T cells. The transfused T cells will also recognise and inactivate the processed HIV in antigen presenting cells.

B. MONOCLONAL ANTIBODIES AGAINST HLA HAPLOTYPES OF DONOR T CELLS

Monoclonal antibodies against the HLA - haplotypes of the allogeneic donor T cells in order to destroy the non - self population and allow the patient to repopulate and re¬ establish the initial original paternal and maternal haplotypes self T cells. By eliminating the non self donor T cells and other allogeneic cells and allowing to the self T cells to be restored, the recognition and processing by antigen presenting cells of HIV and other virus will be normalised and the recurrent HIV or other infectious processes will be controlled.

C. T CELL LINES OF SPECIFIC HLA HAPLOTYPES

T cell lines of specific HLA - haplotypes are developed propagated in vitro and preserved. Banks of T cells of many HLA haplotypes will increase the availability of specific T cells for suitable patients

47 D. GENETICALLY ENGINEERED T CELL LINES WITH GRAFTED MHC I AND MHC II GENES

Genetically engineered T cell lines with grafted MHC I and MHC II genes are established and are compatible to groups of HLA - haplotypes. Grafted T cells that are marked with several MHC I and MHC II antigens are universal in their histocompatibility and are transfused to several groups of patients with corresponding HLA haplotypes.

E. EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION (as in Xenotransplant Project)

The treatment of HIV immune deficient patients with T cell transfusions and monoclonal antibodies will increase the putative T cells and restore the natural immune antiviral and anti allogeneic T cell activity of the host. However after the interruption of the transfusions the allogeneic T cells and the HIV infection can recover and the disease continue. More radically the condition is controlled by the restoration of the thymus that can be restored and re-educated in order to develop normal putative T cells. The restoration of the normal T cell production is achieved by implantation of EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION in the thymus (or in the bone marrow). This preparation will revitalise the thymus, and allow the "nursing" and maturation of thymocytes. T cells will recognise self body cells of the host including putative T cells and also recognise as non "self allogeneic T cells and other donor allogeneic cells and eliminate them. The preparation is the same as the one in the Xenotransplant Project and contains thymus cell lines or genetically engineered cells or crude thymus extracts, implanted in the thymus or bone marrow or in the spleen or injected intravenously or in the thymus artery.

4 8 THEORETICAL APPROACH ON THE EVENTS LEADING TO THE HIV INFECTION

Individuals hosts that are exposed to allogeneic white blood cells, dendritic cells, T cells, thymocytes or other allogeneic cells will interact and recognise the non-self cells through their T cells. The host T cell population after recognising the allogeneic non-self material will destroy by cytotoxicity the donor allogeneic cells.

In the case that the donor T cell manage to survive a dynamic condition will occur that will end with the exhaustion of the specific T cells of the host that recognise the donor T cells. The donor's T cells will continue proliferating and the host's specific T cell population will continue being reduced in population numbers due to the destruction by the donor's T cells. The dynamics of the two T cell populations will determine the clinical condition that will evolve in the individual. In the situation in which the donor T cells are established the individual will become a chimera.

The destruction or deletion of certain T cell clones by the established donor T cells in the host individual will have as a consequence immunologic gaps in the recognition of viral and other antigens on antigen presenting cells. The infection by HIV virus might be in such a situation a consequence of non-response of the individual's T cells to the recognition of HIV processed antigen on the antigen presenting cells. In this theoretical model HIV infection is considered as secondary to the events associated to the introduction and establishment of allogeneic T cells in the host. HIV infection occurring in such allogeneic chimerical individuals will follow the natural history of the disease as described. In parallel and concurrently the individual will be suffering from the consequences of the ongoing pathology of the chimerical condition due to the allogeneic T cells.

4 9 The model can be further complicated in the case of incidental introduction of allogeneic T cells from a third or fourth donor individual.

5 0 4. AUTO IMMUNE PROJECT

SCOPE

The patent describes a novel approach for the treatment of auto immune disease based on the elimination of T cells in the periphery that are auto reactive and the restoration of the proper selection of T cells in the thymus.

Auto immune disease caused by auto reactive cells that were not adequately selected in the thymus, will be the target of this project : aiming to overcome the deficiencies in the negative selection process in the thymus, and to eliminate auto reactive T cells.

RESEARCH AND DEVELOPMENT PRODUCTS AND SYSTEMS

B. T CELL LINES HLA HAPLOTYPES SPECIFIC

C. MHC I AND MHC II GRAFTED T CELL LINES

D. MONOCLONAL ANTIBODIES AGAINST AUTO REACTIVE T CELLS

E. EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION (as in Xenotransplant Project)

5 1 INNOVATIONS AND/OR INVENTIONS DESCRIBED IN THE PATENT

The HLA profile of each patient is evaluated by determining the paternal and maternal initial haplotypes of the subject and determining if the patient has auto reactive T cells From the patient's peripheral blood lymphocytes are separated and sorted and auto reactive T cells collected. T CELLS FROM SYNGENEIC HEALTHY INDIVIDUALS WITH HISTOCOMPATIBLE HLA HAPLOTYPES are tested as to their cytotoxic effect against the putative patient's auto reactive T cells. Cytotoxicity tested in a mixed cell culture system will determine the need for treatment with T CELLS FROM SYNGENEIC HEALTHY INDIVIDUALS WITH HISTOCOMPATIBLE HLA HAPLOTYPES for each patient

T cells from healthy individuals with compatible HLA - haplotypes are transfused in order to initiate a cytotoxic effect against the putative auto reactive T cells of the patient

B. T CELLS LINES HLA HAPLOTYPE SPECIFIC T cell lines of specific HLA - haplotypes are developed propagated in vitro and preserved. Banks of T cells of many HLA haplotypes will increase the availability of specific T cells for suitable patients

C. GENETICALLY ENGINEERED T CELL LINES WITH GRAFTED MHC I AND MHC II GENES

D. MONOCLONAL ANTIBODIES SPECIFIC AGAINST AUTO REACTIVE T CELLS Antibodies against the specific auto reactive T cells of the patients will be administered in order to eliminate this population of T cells that has escaped the negative selection in the thymus. The monoclonal antibodies will be directed against the specific patient's auto reactive T cells and will be therefore

Crude adult histocompatible thymus or cell lines of dendritic cells, epithelial cells, precursor thymocytes, mature thymocytes and other thymus ceils related to the T cell "education" with specific histocompatible HLA - haplotypes and propagated in vitro will be prepared. This syngeneic material or cells will be implanted in the thymus (or in bone marrow) in order to re-establish and enforce the proper negative selection of the developing thymocytes.

E. EARLY HUMAN HLA SPECIFIC THYMUS PREPARATION (as in Xenotransplant

Project)

The preparation will be the same as the one in the Xenotransplant Project. Thymus cell lines or genetically engineered cells are developed in addition to crude thymus

53 extracts. Implantation is made in the thymus or in the bone marrow or in the spleen or is injected intravenously or in the thymus artery. The aim is to restore and re-educate the putative Tcells of the patient in properly selecting T cells and eliminate auto reactive thymocytes.

PATENT IS BASED ON THE FOLLOWING METHODOLOGIES TO BE DEVELOPED:

54

Claims

CLAIMSThis patent is contains four projects describing separately four sets of products and systems related to the application of thymus or thymus cells and related cells and systems that are used in the following conditions :a) organ or tissue transplantation, b) in the treatment of human tumours in patients with defects in immune surveillance and anti tumour cytotoxic effect, c) in the treatment of HIV infection in immune suppressed patients and d) in auto immune disease.

1. XENOTRANSPLANT PROJECT

The combined use of the products and systems described in this patent allow the transplantation and tolerance of organs and tissues from animals to humans or humans to humans.

The claims for new processes that are innovative and presented for the first time for use in immune tolerance of xenotransplants are the following :

1. Transformation at birth to a chimera of the future donor animal to the human histocompatibility group of the recipient human and re-education of the thymus of

55 SHEET the human recipient to produce thymocytes histocompatible to both the human and the donor animal.

2. Establishment of chimerism by the use of two preparations of crude thymus extracts (or of extra corpus propagated thymus cell lines or genetically grafted by MHC I and MHC II genes thymus cell lines).

3. The first of these preparations (EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION) transforms the animal to a histocompatible human HLA specific chimera. The second (THYMUS DONOR INDIVIDUAL PREPARATION) transforms the human recipient to a compatible chimera to the animal donor. 4. Establishment of an outbred or inbred homozygotic or transgenic colony of miniature swine or Pan Troglodytes or other animals (COLONY OF CHIMERICAL ANIMALS) that are chimeras to one or more human histocompatibility haplotypes for use as a source of transplants (XENOTRANSPLANTS FROM GROWN CHIMERICAL HLA - SPECIFIC ANIMALS) to HLA compatible patients. 5. A modification of this project allows the transplantation of allogeneic organs and tissues. The introduction of specific EARLY HUMAN HLA SPECIFIC THYMUS CELL PREPARATION compatible to the HLA specificity of the donor allows the transformation of the recipient to a chimera and thereafter the transplantation of the organ or tissue.

2. HUMANTUMOURIMMUNESUPPRESSIONPROJECT

The combined use of the products and systems described in this patent allow the treatment of tumours in patients that have a defect in the immune surveillance mechanisms or defects in the cytotoxicity effects of T cells. The claims for new processes that are innovative and presented for the first time for use in tumour treatment are the following :

56 ET 1. Identification of the patient's HLA histocompatibility haplotypes

2. Test for the diagnosis of the T cell cytotoxic effect of the putative T cells of the patient and of the T cells to be transfused

3. Transfusion of histocompatible T cells to the cancer patient 4. Establishment of a bank of histocompatible T cells that are propagated in vitro 5. Re-education of the thymus of the patient to restore the production of competent T cells for immune surveillance and for cytotoxic effect by implant of histocompatible thymus or thymus cell lines

The same products and systems are also used in chronic and/or life threatening infectious diseases, whenever immune recognition of infectious agent antigen presenting cells is defective.

3. T CELL IMMUNE DEFICIENCY PROJECT

The combined use of the products and systems described in this patent allow the treatment of HIV in allogeneic patients ( being chimeras to one or more other haplotypes after establishment of the allogeneic T cells). The claims for new processes that are innovative and presented for the first time for use in cancer treatment are the following :

1. Identification of the paternal and maternal histocompatibility haplotypes of the patient as well as the allogeneic histocompatibility group to which the patient has become a chimera to

2. Use of monoclonal specific antibodies to destroy the non putative allogeneic T cells that have been established

3. Transfusion of histocompatible to the putative haplotype group of the patient, T cells

5 7 4. Establishment of a bank of histocompatible T cells that are propagated in vitro

5. Re-education of the thymus of the patient to restore the production of competent T cells for immune surveillance and cytotoxic effect against allogeneic T cells by implant of histocompatible thymus or thymus cell lines

4. AUTO IMMUNE PROJECT

The combined use of the products and systems described in this patent allow the treatment of auto immune disease in patients that have defect in the immune selection mechanisms that by negative selection in the thymus eliminate the auto reactive T cells

The claims for new processes that are innovative and presented for the first time for use in auto immune disease treatment are the following:

1. Identification of the paternal and maternal histocompatibility haplotypes of the patient

2. Use of monoclonal antibodies against auto reactive T cells 3. Transfusion of histocompatible to the HLA of the patient, T cells

4. Establishment of a bank of histocompatible T cells that are propagated in vitro

5. Re-education of the thymus of the patient to restore the elimination process of auto reactive T cells by implant of histocompatible thymus or thymus cell lines

5 8 SUMMARY

Experimental data in the past years in immunology showed that the thymus plays an important role in the selection and development of thymocytes and in the surveillance of "healthy" self cells of the body.

Xenotransplantation (xenogeneic transplantation), surveillance and elimination of tumour cells, HIV infection and immune suppression and auto reactive cells in auto immunity are related to thymus and to the development, "education" or maturation of thymocytes.

The four projects described in this patent are novel methodological applications that are based on the central immunological role of the thymus and of the thymocytes in immune tolerance, in surveillance of cancer cells, in allogeneic tolerance and in the escape of auto reactive T cells in auto immune disease. Using different approaches targeted on the thymus activity - depending to the clinical condition - by restoring, suppressing, enhancing or by-passing this organ, therapeutical effects are aimed in clinical transplantation, cancerology, infectious diseases and auto immune disease.

1. XENOTRANSPLANT PROJECT

59 Transplantation immunology is focused to allogeneic transplants and the methods used are mainly immune suppressive approaches. Xenogeneic transplantation (xenotransplantation) has been investigated to a smaller extent and remains a long term goal. The novel approaches and methods described in this patent refer to four innovative products and systems that are based on the positive and negative thymus selection and to the fact that during embryonic or early life induction of tolerance can be achieved.

The implantation of human thymocytes to new-born donor animals will transform the animal to a chimera of the histocompatible human haplotypes of the implant.

Extraction of thymus cells after a few days or weeks and preservation will allow to prepare material that will be used prior to xenotransplantation to the human recipient patient. The patient by being implanted with the thymus of the animal donor will re¬ educate the patient's thymus to positively and negatively select thymocytes histocompatible to both the human self and to the animal donor. The human recipient patient will be rendered a chimera to the donor animal and the thymus donor cells will be tolerated and recognised as self by the recipient. As a result to this, the xenotransplantation of animal organs or tissues belonging to the same individual animal to which the human recipient has become a chimera to, will be tolerated and no rejection will occur.

With these methods, products and systems double self recognition is achieved : recognition of transplant as self by human patient recipient and recognition as self of recipient by xenotransplant.

Crude thymus or cell lines of thymus cells or genetically engineered thymus cell lines are described. The method applies to both outbred or inbred homozygotic donor animal colonies of miniature swine or Pan Troglodytes. Animals from an established colony of transgenic to one or more human MHC I and MHC II genes are used in an

60 equivocal manner as the inbred homozygotic and outbred animals. The use of transgenic homozygotic animal strains to one or more MHC I and MHC II genes allows direct xenotransplantation to corresponding HLA specificity human recipients. The use of thymus cell lines or genetically engineered thymus cell line is aiming in reducing the number of HLA specific preparations and obtaining products that group more than one HLA specificities. The combined use of inbred homozygotic animals and grafted with MHC I and MHC II thymus cell lines allows improved tolerance and reduced number of HLA specific products therefore broader applicability. The use of human new-bom thymus cells to human recipients allows allogeneic transplantation. The thymus cells introduced are histocompatible to the HLA of the donor human.

2. TUMOUR IMMUNE SUPPRESSION PROJECT

The patent describes a novel and innovative approach consisting of four products and systems proposed for the treatment of cancers that are due to a defect in the immune surveillance mechanisms or to a defect in the Tcells that are responsible for a cytotoxic effect on tumour cells. A diagnostic histocompatibility typing test for the cancer patient to determine the paternal and maternal haplotypes is used before treatment.

The patent describes a treatment consisting of administration of histocompatible T cells from healthy donors or of histocompatible T cell lines or of genetically engineered T cells. The described treatment is aiming to re-establish the immune surveillance system and to enforce the cytoxic effects of T cells. The method applies to the use of the T cells alone or in combination to surgical, chemotherapeutic or radiotherapeutic approaches. The method is expanded to the

5 1 use of the thymus cell preparation described in the Xenotransplant Project. The implantation of healthy new-born histocompatible thymus cells will allow the re¬ education of the patient's thymus in restoring the defected T cell development in the thymus and re-establishing the immune surveillance.

In addition to the TUMOUR IMMUNE SUPPRESSION PROJECT described in this patent, restoration of thymus activity can be envisaged in infectious diseases where deficient immunological functions are deficient (i.e. antiviral, antibacterial, antifungal or antiparasitic immune activity). In such cases therapeutic thymus preparations (i.e. healthy individual T ceils, T cell lines HLA -haplotype specific) provide therapeutic effects.

3. T CELL IMMUNE DEFICIENCY PROJECT

The products and systems described in this patent are based on an unpublished and original explanation of HIV infection and aim in the treatment of HIV infection in immune deficient patients.

HIV infection is considered as secondary after an allogeneic introduction of white blood cells or other allogeneic cells from one individual to another by different vehicles (i.e. sperm, blood). If such allogeneic cells manage to be tolerated then the individual becomes a chimera to the donor individual of the allogeneic cells. As a consequence the chimerical individual will develop gaps in the immune response of T cells due to exhaustion or elimination of several putative T cell clones.

The HIV secondary infection and the entire immune deficiency condition is treated by a novel approach consisting of five products and systems. The treatment is aiming in

6 2 identifying the histocompatibility group of the patient and of the established donor T cells and the administration of antibodies to eliminate the donor T cells and of histocompatible "healthy" T cells to restore-the immune deficiency.

The method is expanded to the use of the thymus cell preparation described in the Xenotransplant Project. The implantation of "healthy" new-bom histocompatible thymus cells will allow the re-education of the patient's thymus in restoring the defected T cell development in the thymus and re-establishing the immune surveillance as to self recognition.

4. AUTO IMMUNE PROJECT

The patent describes a novel method for treatment of auto immune disease caused by a defect in the negative selection in the thymus that permits the maturation and circulation in the blood of auto reactive T cells.

The patent describes five products and systems aiming in the diagnostic identification of the patient's histocompatibility group, in the destruction of the circulating auto immune T cells and in the restoration of the negative selection in the thymus and the re-establishment in the thymus of a correctly selected thymocyte population.

In addition to the auto immune disease, restoration of thymus deficient activity can be envisaged in hypersensibilities. In such cases therapeutic thymus preparations (i. e. healthy individuals T cells, T cell lines HLA - haplotype specific ) provide therapeutic effects.

6 3