Lymphodepletion by selective apoptosis induction in t cells for immune therapy applications

Abstract

Compositions and methods are provided for improved T cell lymphodepletion in a mammalian host, using a conditioning regimen wherein concomitant treatment of an effective dose of an antibody specific for CD3 and an effective dose of a BCL2 inhibitor provides for improved T cell depletion. The conditioning regimen may further comprise administering an immunosuppressive treatment or agent.

Description

[0001] LYMPHODEPLETION BY SELECTIVE APOPTOSIS INDUCTION IN T CELLS FOR IMMUNE THERAPY APPLICATIONS

[0002] GOVERNMENT SUPPORT

[0003]

[0001] This invention was made with Government support under contract R01 (1 R01 DK132549), SPO: 229763 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005]

[0002] This application claims priority to the filing of United States Provisional Application Serial No. 63 / 749,938, filed January 27, 2025, the disclosure of which application is herein incorporated by reference.

[0006] BACKGROUND

[0007]

[0003] The success of autologous and allogeneic T cell-based immunotherapy often depends on robust depletion of recipient lymphocytes prior to adoptive transfer. This is in large part because recipient T cells can prevent the homeostatic expansion of donor T cell therapies or reject allogeneic products. This has been well described in hematopoietic stem cell transplantation (HSCT), where recipient T cells increasingly prevent donor engraftment upon reduction in the intensity of chemo-therapy and radiation-based conditioning, unless immune agents like antithymocyte globulin (ATG) are specifically used for T cell depletion. Unfortunately, ATG is commonly associated with significant infusion reactions, late-term serum sickness, higher infection risk, and increased nonrelapse mortality in some settings. ATG also has a long half-life and is highly variable in its clearance, complicating its use. Importantly, residual ATG at the time of adoptive T cell therapy product administration can eliminate or impair the product, making ATG impractical for use with advanced T cell therapies such as CAR T cells.

[0008]

[0004] Chemotherapy-based agents like cyclophosphamide, fludarabine, or bendamustine are commonly used for T cell depletion. In HSCT, posttransplant cyclophosphamide (PTCy) has proven effective in preventing graft-versus-host disease (GVHD). Although PTCy is generally well tolerated, it can lead to organ toxicity in older patients orthose with significant comorbidities, or for patients with immunodeficiency, red cell dyscrasias, or auto-immune conditions in which chemotherapy is not generally utilized, but who still may derive great clinical benefit from T cell immunotherapies. For immune tolerance to organ or tissue grafts such as pancreatic islets, effective T cell depletion is critical, especially when combining tissue transplant with HSCT. This approach promotes sustained mixed hematopoietic chimerism, allowing immunosuppressionwithdrawal. Current clinical protocols of combined organ or tissue transplantation depend almost entirely upon ATG or PTCy and can present similar challenges to patients receiving HSCT alone.

[0009]

[0005] There is a clinical need to improve the depletion of T cells to create safer and more effective therapies.

[0010] SUMMARY OF THE INVENTION

[0011]

[0006] Compositions and methods are provided for improved T cell lymphodepletion in a mammalian host, using a conditioning regimen wherein concomitant treatment of an effective dose of an antibody specific for CD3 and an effective dose of a BCL2 inhibitor provides for improved T cell depletion. The treatment induces T cell apoptosis, which can trigger changes that improve immune tolerance, including an increase in the number of one or more of regulatory (Treg) cells, myeloid derived suppressor cells (MDSC), and tolerogenic dendritic cells (DC2). The regimen finds use, for example, in conditioning a recipient for transplantation of hematopoietic stem cells (HSCT), for solid organ transplantation, for transplantation of chimeric antigen receptor T cells (CAR-T), for treatment or prevention of graft versus host disease (GVHD), and the like.

[0012]

[0007] The conditioning regiment resets the immune system and promotes tolerance. This approach effectively overcomes the T cell-mediated engraftment barrier and leads to the induction of tolerogenic cell subsets, including an increase of regulatory cells, regulatory T cells, tolerogenic CD8 SIRPa+dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs). In an islet transplantation model, the conditioning regimen reduces alloreactivity, facilitates the persistence of engrafted donor cells, results in the establishment of chimerism and the long-term islet survival.

[0013]

[0008] In some embodiments the conditioning regimen is combined with an immunosuppressive regimen. In some embodiments the immunosuppressive regimen comprises administration of an effective dose of an antibody specific for TIM1. In some embodiments the immunosuppressive regimen comprises administration of total lymphoid irradiation. In some embodiments the immunosuppressive regimen comprises administration of a low dose total body irradiation. In some embodiments the administration is performed in the absence of anti-thymocyte globulin (ATG).

[0014]

[0009] In some embodiments the antibody specific for CD3 specifically binds to human CD3.

[0015] In some embodiments the antibody specific for CD3 lacks an Fc region sequence. In some embodiments the antibody specific for CD3 comprises an Fc sequence modified to reduce binding to high affinity FcyR. In some embodiments the antibody specific for CD3 is an F(ab)2fragment, or an F(ab’)2 fragment. In some embodiments the anti-CD3 antibody is a fragment or derivative of muromonab-CD3, otelixizumab, teplizumab, foralumab or visilizumab.

[0016]

[0010] In some embodiments the anti-CD3 antibody or anti-CD3 Fab fragment is a part of a bivalent or trivalent molecular targeting complex, including BiTEs, with examples such as blinitobumab or others.

[0017]

[0011] BCL-2 inhibitors target the anti-apoptotic protein B-cell lymphoma 2 (BCL-2), which plays a critical role in cell survival by inhibiting apoptosis. In some embodiments the BCL2 inhibitor is venetoclax (ABT-199), a selective small-molecule inhibitor approved for treating chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). In other embodiments the BCL2 inhibitor is navitoclax (ABT-263); S55746 (BCL201); Obatoclax (GX15-070); gossypol, etc.

[0018]

[0012] In some embodiments the conditioning regimen of the disclosure is administered prior to bone marrow transplantation. In some embodiments the conditioning regimen of the disclosure is administered prior to hematopoietic stem cell transplantation. In some embodiments the conditioning regimen of the disclosure is administered prior to CAR-T cell administration. In some embodiments the conditioning regimen of the disclosure is administered prior to solid organ transplantation, where the solid organ transplantation is optionally combined with BMT or HSCT. In some embodiments the conditioning regimen of the disclosure is administered for the treatment of GVHD, where administration may be prior to, or following transplantation of hematopoietic cells. In some such embodiments administration follows a determination of GVHD. In some embodiments the conditioning regimen of the disclosure is administered for the treatment of an autoimmune disease. The methods allow engraftment to treat hematologic disorders, to reduce undesirable T cell reactivity and to tolerize a recipient to a donor-type HLA for organ transplantation.

[0019]

[0013] In some embodiments, the methods described herein may comprise the steps of: HLA typing a donor and recipient to determine an HLA-matched or HLA- mismatched pair; obtaining hematopoietic cells and / or an organ for transplantation from the donor; treating the recipient with the conditioning regimen of the disclosure, and monitoring the recipient for engraftment. The methods described herein apply to both HLA-matched and HLA-mismatched transplantation conditions, for example HLA-mismatched and not haploidentical transplantations, haploidentical transplantations; etc. In some embodiments the recipient is immunocompetent. The administration of the pre-transplantation conditioning regimen is repeated as necessary to achieve the desired level of ablation. Following transplantation with donor stem cells, the recipient may be a chimera or mixed chimera for the donor cells.

[0020]

[0014] The methods of the invention are also useful in the induction of tolerance in a patient, for example tolerance to donor tissue, e.g. in organ transplants; tolerance to autoantigens,e.g. in the context of treatment of autoimmune disease; and the like. In one embodiment of the invention, a method is provided for inducing tolerance in a patient, comprising administering to a patient a conditioning regimen of the disclosure. Following the conditioning regimen, the recipient is optionally infused with an effective dose of hematopoietic stem and progenitor cells, thereby providing immune tolerance to the donor cells for organ transplants.

[0021] BRIEF DESCRIPTION OF THE FIGURES

[0022]

[0015] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0023]

[0016] FIGS. 1A-1 E. Venetoclax (Ven) and anti-CD3 (aCD3) F(ab’)2 treatment enhances in vitro apoptosis, T cell depletion, and promotes regulatory T cells (Tregs) and tolerogenic cell subsets in vivo. (A) Experimental scheme of the in vitro apoptosis assay. Splenocytes were isolated from B6 mice and cultured at rest for 48 hours in the presence of Ven (500 nM), aCD3 F(ab)2 (1 pg / well), and aTIM-1 (20 mg / mL). (B) Increased T cell early apoptosis and (C) depletion of T cells were observed after 48 hours of in vitro culture in the presence of Ven, aCD3 F(ab)2, and aTIM-1 . Absolute counts (ABS) of T cells, CD4+ T, CD8+ T, and Tregs, as well as the ratio of ABS of Treg / CD4+, Treg / CD8+, T cells naive CD8+ / CD8+ T cells, and effector memory CD8+ / CD8+ T cells are shown (n = 4-6 / group). Pooled data from 3 independent experiments are shown. For statistical analysis, the 1-way analysis of variance (ANOVA) test was used (*P <.05,**P < .01 ,***P < .001 ,****P < .001 ). (D) Experimental scheme of in vivo, Ven, aCD3 F(ab)2, and aTIM-1 conditioning regimen. B6 mice were conditioned with either aCD3 monoclonal antibodies (mAbs) (50 pg / mouse) on day -5 alone, aCD3 F(ab)2 (50 pg / mouse) alone on day -6 to day -1 , a combination of Ven (100 mg / kg) on day -3 and day -2, and aCD3 F(ab)2, or a triple combination of Ven, aCD3 F(ab)2, and aTIM-1 (400 pg / mouse) on day -4 and day -1 . (E) ABS of T cells, CD4+ T, CD8+ T, and Tregs, as well as the ratio of ABS of T reg / CD4+, T reg / CD8+ T cells in the spleen and bone marrow (BM) on day 0 post-Ven; aCD3 F(ab)2 and aTIM-1 conditioning are shown (n = 7-8 / group). Pooled data from 2 independent experiments are shown. For statistical analysis, the 1-way ANOVA test was used (*P < .05,**P < .01 ,*** P < ,001 ,****P < .001). Error bars indicate standard error of the mean (SEM). TIM-1 , T cell immunoglobulin and mucin 1 .

[0024]

[0017] FIGS. 2A-2F. Venetoclax (Ven) and anti-CD3 (aCD3) F(ab’)2conditioning combined with total lymphoid irradiation (TLI) enhances CD8+T cell depletion, regulatory T cell (Treg) expansion, and sustained mixed chimerism following bone marrow transplantation (BMT). (A)Experimental scheme of in vivo Ven, antithymocyte globulin serum (ATS), aCD3 F(ab)2, aTIM- 1 , and TLI / ATS conditioning regimen. B6 mice were conditioned with either ATS alone (5 doses), aCD3 F(ab)2(50 pg / mouse) alone on day -6, a combination of Ven on day -3 and day -2 (100 mg / kg), and aCD3 F(ab)2 on day -6 to day -1 , or a triple combination of Ven, aCD3 F(ab)2, and aTIM-1 (400 pg / mouse) on day -4 and day -1. All animals also received 10 doses of TLI (250 cGy). (B) Cell subsets in the blood on day 0 (n = 5-10 / group) post-BMT. Pooled data from 2 independent experiments are shown. For statistical analysis, the 1-way ANOVA test was used (*P < .05,**P < ,01,***P < ,001,****P < .001). (C) Time course of CD90.2+ T cells, CD8+ T cells, CD4+ T cells, and Treg cells in the blood (n = 5-10 / group). Pooled data from 2 independent experiments are shown. For statistical analysis, the 2-way ANOVA test was used (*P < .05,**P < ,01,***P < ,001 ,****P < .001). (D) Time course of mixed chimerism levels of total, T cells, B cells, and myeloid cells (n = 5-10 / group). For statistical analysis, the 2-way ANOVA mixed test was used (*P < .05,**P < .01 ,***P < .001 ,****P < .001). Error bars indicate SEM. (E) Mixed chimerism levels of total, T cells, Tregs, B cells, and myeloid cells on day 90 in the bone marrow (BM) (n = 5-10 / group) post-BMT. Pooled data from 2 independent experiments are shown. For statistical analysis, the 1-way ANOVA test was used *P < ,05,**P < .01 ,***P < .001 ,****P < .001). (F) Mixed chimerism levels of total, T cells, Tregs, B cells, and myeloid cells on day 90 in the spleen (n = 5-10 / group) post-BMT. Pooled data from 2 independent experiments are shown. For statistical analysis, the 1 -way ANOVA test was used *P < .05,** P < ,01 ,***P < ,001 ,****P < .001). Error bars indicate SEM. TIM-1 , T cell immunoglobulin and mucin 1.

[0025]

[0018] FIGS. 3A-3D. Combination of venetoclax (Ven) and anti-CD3 (aCD3) F(ab’)2 conditioning with low-dose TBI enhances lymphodepletion and promotes immunoregulatory cell subsets, resulting in persistent mixed chimerism following bone marrow transplantation (BMT). (A) Experimental scheme of in vivo Ven, aCD3 F(ab)2, aTIM-1 , and low-dose nonmyeloablative TBI conditioning regimen. B6 mice were conditioned with either antithymocyte globulin serum (ATS) alone on day -7 and day -5, aCD3 monoclonal antibodies (mAbs) (50 pg / mouse) on day -5, aCD3 F(ab)2 (50 pg / mouse) alone on day -6 to day -1 , a combination of Ven (100 mg / kg) on day -3 and day -2 and aCD3 F(ab)2, or a triple combination of Ven, aCD3 F(ab)2, and aTIM-1 (400 pg / mouse) on day -4 and day -1. All animals also received low-dose TBI (250 cGy). (B) Cell subsets in the blood on day 14 (n = 5- 9 / group) post-BMT. Pooled data from 2 independent experiments are shown. For statistical analysis, the 1-way ANOVA test was used (*P < .05, **P <.01 , ***P < .001). (C) Mixed chimerism levels of total, T cells, B cells, regulatory T cells (Tregs), myeloid, and granulocytes on day 14 (n = 5-9 / group) post-BMT. Pooled data from 2 independent experiments are shown. For statistical analysis, the 1-way ANOVA test was used (*P < .05, **P < .01 ,***P < .001). (D)Time course of mixed chimerism levels of total, T cells, B cells, Tregs, myeloid, and granulocytes (n = 4-9 / group). For statistical analysis, the multiple unpaired t test was used (*P < .05, **P < .01). Error bars indicate SEM. TIM-1 , T cell immunoglobulin and mucin 1.

[0026]

[0019] FIGS. 4A-4E. Venetoclax (Ven), and anti-CD3 (aCD3) F(ab’)2 conditioning induced sustained mixed chimerism and immune tolerance to allogeneic islets. (A) Experimental scheme of the conditioning regimen and experimental design of islet and bone marrow transplantation (BMT). B6 mice were conditioned with either aCD3 F(ab)2 (50 pg / mouse) alone on day -6 to day -1 , a combination of Ven (100 mg / kg) on day -3 and day -2 and aCD3 F(ab)2, or a triple combination of Ven, aCD3 F(ab)2, and aTIM-1 (400 pg / mouse) on day -4 and -1 . All animals also received low-dose TBI (250 cGy). On day 0, the mice received 30 x 106allogeneic BALB / c bone marrow (BM) cells, and on day 1 , 100 firefly luciferase-expressing BALB / c islets were transplanted under the left kidney capsule. (B) Cell subsets in the blood (BL) on day 14 (n = 5-13 / group) and day 30 (n = 8-17 / group) post-BMT. Pooled data from 3 independent experiments are shown. (C) Sustain levels of mixed chimerism on day 30 (n = 8- 17 / group) post-BMT. Pooled data from 3 independent experiments are shown. For statistical analysis, the 1-way ANOVA test was used (*P < .05, **P < ,01,***P < ,001 ,***P <.0001). (D) Representative of bioluminescence imaging (BLI) of luc+ transplanted islets on day +90 postislet transplantation. (E) Bioluminescence signal intensity time course of firefly luciferaseexpressing transplanted islets, pooled across 3 independent experiments (n = 6-12 / group). For statistical analysis, the multiple unpaired t test was used (*P < .05,**P < .01). Error bars indicate SEM. Ctl, control; TIM-1, T cell immunoglobulin and mucin 1.

[0027]

[0020] FIGS. 5A-5G. Venetoclax (Ven), and anti-CD3 (aCD3) F(ab’)2 treatment following allogeneic bone marrow transplantation (BMT) prevents graft-versus-host disease (GVHD). (A) Experimental scheme of a fully major histocompatibility complex (MHC) mismatched BMT model, PT-aCD3 Fab + Ven treatment. BALB / c mice received allogeneic T cell-depleted (TCD) bone marrow (BM) and T cells from MHC major mismatched B6 donors. T cells were isolated from DTR-Foxp3+Luc+GFP+ B6 mice, allowing for bioluminescence imaging (BLI). On days 3 and 4 posttransplant, the mice were treated with either the combination of Ven (100 mg / kg) and aCD3 F(ab’)2 (50 pg / mouse), Ven alone, or aCD3 F(ab’)2 alone. (B) Overall survival, and (C) GVHD score of allogeneic transplanted BALB / c recipient mice after BMT (n = 5 / group). Representative data are shown from 2 independent experiments. The log-rank test was used for statistical evaluation of mouse survival (Kaplan-Meier survival curves) with comparison of conventional T cells (Tcons) + Fab + Ven and Tcons + PTCy to Tcons shown (*P < .05). Statistical analysis for GVHD score is shown between Tcons + Fab + Ven and Tcons groups (multiple unpaired Student’s t test) *P < .05, **P < .01. (D) Representative BLI of allogeneic transplanted BALB / c recipient mice after BMT of Foxp3+Luc+ cells on day 6 andday 80 post-BMT. (E) Bioluminescence signal intensity of Foxp3+Luc+ cells on day 6 and day 80, pooled across 2 independent experiments (n = 8 / group). For statistical analysis, the 1-way ANOVA test was used (*P < .05, **P < .01). (F) Percentage of CD4+CD25+GFP+ cells is shown in the blood of allogeneic transplanted BALB / c recipient mice on day 6 and day 21 post-BMT, and (G) ratio of CD4+CD25+ / Tcons in the blood on day 6 post-BMT. Pooled data from 2 independent experiments are shown (n = 6-10 / group). For statistical analysis, the 1- way ANOVA test was used (*P < .05, **P < .01 ). Error bars indicate SEM. PTCy, posttransplant cyclophosphamide.

[0028]

[0021] FIGS. 6A-6F. Venetoclax (Ven) and anti-CD3 (aCD3) F(ab’)2 treatment following bone marrow transplantation (BMT) protects against graft- versus-host disease (GVHD) and exhibits reduced toxicity and inflammatory response compared to posttransplant cyclophosphamide (PTCy). (A) Experimental scheme of a fully major histocompatibility complex (MHO) mismatched BMT model, PT-aCD3 Fab + Ven and PTCy treatment. BALB / c mice received allogeneic TCD-BM and T cells from MHC major mismatched B6 donors. On days 3 and 4 posttransplant, the mice were treated with either the combination of Ven (100mg / kg) and aCD3 F(ab’)2 (50 pg / mouse), aCD3 F(ab’)2 alone, or cyclophosphamide (50 mg / kg). (B) Overall survival (C) GVHD score of allogeneic transplanted BALB / c recipient mice after BMT (n = 5 / group). Representative data are shown from 2 independent ex-periments. The log-rank test was used for statistical evaluation of mouse survival (Kaplan-Meier survival curves) with comparison of conventional T cells (Tcons) + Fab + Ven and Tcons + PTCy to Tcons shown (*P < .05). Simple linear regression analysis was performed for both GVHD and weight loss with comparison of Tcons + Fab + Ven and Tcons + PTCy to Tcons shown (*P < .05, ***P < .001). (D) Percentage of lymphocytes, CD90.2+, CD8+ T cells, and the ratio of CD4+CD25+ / CD4+ are shown in the blood of allogeneic transplanted BALB / c recipient mice on day 6 post-BMT. Pooled data from 2 independent experiments are shown (n = 6-10 / group). For statistical analysis, the 1 -way ANOVA test was used (*P < .05, **P < .01 , ***P < .001 , ****P < .0001). (E) Cytokine and chemokine levels in the serum of allogeneic transplanted BALB / c recipient mice, as assessed by a multiple assay (Luminex). A total of 26 cytokines and chemokines were analyzed. Significant differences between groups were detected for interleukin (IL)-2, MIP-1a, IL-22, MIP-ip, and IL-6. For the remaining analytes, there was either no statistical difference between the groups or the cytokine / chemokine levels were below the assay’s limit of detection. The dotted line represents the Luminex detection limit. For statistical analysis, the 1 -way ANOVA test was used; *P < .05, **P < .01. (F) Serum levels of creatine, alanine transaminase (ALT), and troponin as a measure of kidney, liver, and heart toxicity. For statistical analysis, the 2-tailed Student’s t test was used. *P < .05. Error bars indicate SEM. BM, bone marrow.

[0022] FIGS. 7A-7E. Comparison of posttransplant depletion strategies for islet allograft tolerance with full donor chimerism. (A) Schematic of the conditioning regimen and experimental design of combined islet and bone marrow transplantation (BMT) with PT-aCD3 Fab + Ven and posttransplant cyclo-phosphamide (PTCy) treatment. B6 mice were conditioned with either anti-CD3 (aCD3) F(ab’)2 (50 pg / mouse) alone, a combination of venetoclax (Ven) (100 mg / kg) and aCD3 F(ab’)2, or a triple combination of Ven, aCD3 F(ab’)2, and aTIM-1 (400 pg / mouse) with low-dose TBI (250 cGy). On day 0, the mice received 50 x 106 allogeneic BALB / c bone marrow cells, and on day 1 , 100 firefly luciferase-expressing BALB / c islets were transplanted under the kidney capsule. A single dose of aCD3 F(ab’)2 on day 3, and Ven on day 3 and 4, or 2 doses of cyclophosphamide (50 mg / kg) on days 3 and 4 was administered. (B) Representative bioluminescence imaging (BLI) of firefly luciferaseexpressing islets on day +75 post-islet trans-plantation. (C) Bioluminescence signal intensity time course of firefly luciferase-expressing (luc+) islets, pooled across 2 independent experiments (n= 8 mice / group). (D) Time course of total chimerism (upper panel) and T cell chimerism (bottom panel) in the blood of B6 recipient mice transplanted with BALB / c BM cells. (E) Chemokine levels in the serum of mice as assessed by multiplex assay (Luminex) on day 75 post-islet transplantation. The dotted line represents the Luminex detection limit. For statistical analysis, the 2-tailed Student’s t test was used. *P < .05. Error bars indicate SEM. Ctl, control; TIM-1 , T cell immunoglobulin and mucin 1 .

[0029]

[0023] FIGS. 8A-8D. Venetoclax and aCD3 F(ab’)2treatment affects naive and central memory T cells and enhances DC2 cell subsets. (A) Increased ratio SIRPa+ / CD8+T cell in the venetoclax, aCD3 F(ab’)2and aTIM-1 group in vitro. ABS SIRPcr7CD8+T cell ration, ABS of MDSCs and ABS of B220+cells in the venetoclax, aCD3 F(ab’)2and aTIM-1 treated group (n=4-6 / group). Pooled data of three independent experiments are shown. For statistical analysis the one-way ANOVA test was used (*p<0.05) (B) Reduced percentage of naive and central memory CD8+T cells in the spleen and BM on dayO post venetoclax, aCD3 F(ab’)2 and aTIM-1 conditioning in vivo (n=7-8 / group). (C) Increased ratio of SIRPa+ / CD8+T and (D) ABS of B cells in the spleen and BM on dayO post venetoclax, aCD3 F(ab’)2 and aTIM-1 conditioning in vivo. Pooled data of two independent experiments are shown. For statistical analysis the one-way ANOVA test was used (*p< 0.05). Error bars indicate SEM.

[0030]

[0024] FIGS. 9A-9D. Venetoclax and aCD3 F(ab’)2conditioning combined with TLI enhances effector memory CD8+T cells in the BL, CD8+T Cell depletion, and sustained mixed chimerism following BMT in the spleen and BM. (A) Percentage of MDSCs, SIRPcr cells, CD11b+cells and (B) naive and memory T and CD8+T cells in the peripheral blood on dayO post in vivo TLI / ATS, aCD3 F(ab’)2 alone, aCD3 F(ab’)2 plus venetoclax and aCD3 F(ab’)2 plus venetoclax and aTIM-1 and conditioning regimen (n=5-10 / group). (C) Time course ofpercentage of B220+, CD11b+, SIRPa+cells and MDSCs in the blood on dayO (n=5-10 / group). Pooled data of two independent experiments are shown. For statistical analysis the two-way ANOVA test was used (*p < 0.05,**p< 0.01 ,***p< 0.001 ). (D) Donor chimerism of MDSCs and SIRPa-i- cells in the spleen and BM d90 post BMT (n=5-10 / group). Pooled data of two independent experiments are shown. For statistical analysis the one-way ANOVA test was used *p < 0.05,**p< 0.01 ***p< 0.001 ,****p< 0.001). Error bars indicate SEM.

[0031]

[0025] FIG. 10. Combination of venetoclax and aCD3 F(ab’)2conditioning with low-dose TBI enhances lymphodepletion and promotes immunoregulatory cell subsets, resulting in persistent mixed chimerism following BMT. Mixed chimerism levels of total, T cells, B cells, Treg, and DC2 in the spleen and BM on day30 (n=5 / group) post BMT. For statistical analysis the one-way ANOVA test was used (*p< 0.05). Error bars indicate SEM.

[0032]

[0026] FIG. 11. Venetoclax, and aCD3 F(ab’)2treatment following allogeneic BMT prevents GVHD. Weight loss of allogeneic transplanted BALB / c recipient mice after BMT (n=5 / group). Representative data are shown from two independent experiments. Statistical analysis for GVHD score is shown between Tcons+Fab+Ven and Tcons groups (multiple unpaired Student’s t test). *p<0.05,**p<0.01. Error bars indicate SEM.

[0033]

[0027] FIG. 12. PT-aCD3 Fab+Ven and PTCy treatment following allogeneic BMT prevents GVHD. Weight loss of allogeneic transplanted BALB / c recipient mice after BMT treated with either PT-aCD3Fab+Ven and PTCy (n=5 / group). Representative data are shown from two independent experiments. Statistical analysis for GVHD score is shown between Tcons+Fab+Ven and Tcons groups and Tcons+PTCy (Simple linear regression analysis) *p<0.05,***p<0.01. Error bars indicate SEM.

[0034] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035]

[0028] It is to be understood that this invention is not limited to the particular methodology, products, apparatus and factors described, as such methods, apparatus and formulations may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by appended claims.

[0036]

[0029] It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a drug candidate" refers to one or mixtures of such candidates, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[0037]

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this inventionbelongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing devices, formulations and methodologies which are described in the publication and which might be used in connection with the presently described invention.

[0038]

[0031] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0039]

[0032] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

[0040]

[0033] Generally, conventional methods of protein synthesis, recombinant cell culture and protein isolation, and recombinant DNA techniques within the skill of the art are employed in the present invention. Such techniques are explained fully in the literature, see, e.g., Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982); Sambrook, Russell and Sambrook, Molecular Cloning: A Laboratory Manual (2001); Harlow, Lane and Harlow, Using Antibodies: A Laboratory Manual: Portable Protocol No. I, Cold Spring Harbor Laboratory (1998); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; (1988).

[0041]

[0034] "Concomitant administration" of active agents in the methods of the invention means administration with the reagents at such time that the agents will have a therapeutic effect at the same time. Such concomitant administration may involve concurrent ( / .e. at the same time), prior, or subsequent administration of the agents. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention.

[0042]

[0035] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In oneembodiment, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0043]

[0036] As used herein, the term “isolated” means material that is substantially or essentially free from components that normally accompany it in its native state. In particular embodiments, the term “obtained” or “derived” is used synonymously with isolated.

[0044]

[0037] The terms “subject,” “patient” and “individual” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. A “subject,” “patient” or “individual” as used herein, includes any animal that exhibits pain that can be treated with the vectors, compositions, and methods contemplated herein. Suitable subjects (e.g., patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

[0045]

[0038] As used herein “treatment” or “treating,” includes any beneficial or desirable effect, and may include even minimal improvement in symptoms. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0046]

[0039] As used herein, “prevent,” and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of a symptom of disease. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and / or burden of disease prior to onset or recurrence.

[0047]

[0040] As used herein, the term “amount” refers to “an amount effective” or “an effective amount” is the dose of a cell population that achieves a beneficial or desired prophylactic or therapeutic result, including clinical results.

[0048]

[0041] A “therapeutically effective amount” of a cell population may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the virus or cell to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or cell are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient).

[0049]

[0042] An “increased” or “enhanced” amount of a physiological response, e.g. cytotoxicity against a target cell, is typically a “statistically significant” amount, and may include anincrease that is 1.1 , 1 .2, 1 .5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1 , e.g., 1.5, 1.6, 1.7.

[0050] 1 .8, etc.) the level of activity in an untreated cell.

[0051]

[0043] A “decrease” or “reduced” amount of a physiological response, e.g. cytotoxicity against a target cell, is typically a “statistically significant” amount, and may include an decrease that is 1.1 , 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1 , e.g., 1.5, 1 .6, 1.7. 1.8, etc.) the level of activity in an untreated cell.

[0052]

[0044] By “maintain,” or “preserve,” or “maintenance,” or “no change,” or “no substantial change,” or “no substantial decrease” refers generally to a physiological response that is comparable to a response caused by either vehicle, or a control molecule / cell composition. A comparable response is one that is not significantly different or measurable different from the reference response.

[0053]

[0045] The term “exogenous” is used herein to refer to any molecule, including nucleic acids, protein or peptides, small molecular compounds, and the like that originate from outside the organism. In contrast, the term “endogenous” refers to any molecule that originates from inside the organism (i.e., naturally produced by the organism).

[0054]

[0046] CD3. The CD3 protein, also known as the T-cell receptor complex, is a crucial component of the T-cell receptor (TCR) signaling pathway, and plays a critical role in transmitting signals from the TCR to the interior of the T cell, triggering a series of intracellular events that lead to T-cell activation. The CD3 protein complex consists of five distinct polypeptide chains, designated as CD3y, CD35, CD3E, and CD3^. These chains assemble together to form two functional dimers: CD3yc and CD36E. These dimers, along with the CD3^ chain, are associated with the TCR complex. The CD3y, CD35, and CD3E chains are immunoglobulin-like molecules, while the CD3^ chain is more structurally diverse.

[0055]

[0047] The extracellular region of CD3 proteins contains immunoglobulin-like domains, responsible for interacting with the TCRa|3 heterodimer. These interactions contribute to the stabilization of the TCR complex and facilitate antigen recognition. The cytoplasmic tail of the CD3 subchains contain immunoreceptor tyrosine-based activation motifs (ITAMs), which are phosphorylated upon TCR engagement, initiating downstream signaling events.

[0056]

[0048] The prototypical monospecific anti-CD3 antibody is muromonab-CD3 (OKT3), a murine lgG2a antibody historically used to treat acute allograft rejection by inducing profound T-cell activation followed by depletion and anergy; although effective, its use has largely been discontinued due to immunogenicity and severe cytokine release-related toxicities. More recently, teplizumab, a humanized, Fc-receptor-nonbinding anti-CD3s antibody, has gainedclinical approval for delaying the onset of type 1 diabetes in high-risk individuals by inducing partial T-cell exhaustion and immune tolerance without extensive T-cell depletion. Other monospecific anti-CD3 antibodies such as otelixizumab, visilizumab, and foralumab have been evaluated in autoimmune and inflammatory diseases. In oncology, CD3 is most prominently leveraged as one arm of bispecific T-cell-engaging antibodies, including approved agents such as blinatumomab (CD19xCD3) for B-cell acute lymphoblastic leukemia, tebentafusp (gp100xCD3) for uveal melanoma, and newer molecules like mosunetuzumab, glofitamab, epcoritamab, and teclistamab, which pair CD3 with B-cell or plasma cell antigens. In these settings, the anti-CD3 component is essential for recruiting and activating cytotoxic T cells at the site of disease, making CD3 targeting a cornerstone of modern immunotherapy despite the need for careful management of cytokine-mediated toxicities. In some embodiments, the antibody teplizumab is used in the methods of the disclosure.

[0057]

[0049] The dose of an anti-CD3 antibody, e.g. teplizumab, in a human recipient may be from about 1 to about 1000 mg / day, e.g. at least about 1 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, and up to about 400 mg, 600 mg, 800 mg, 1000 mg or more.

[0058]

[0050] BCL2 inhibitor. Inhibitors of B-cell lymphoma 2 (BCL-2) are a class of targeted therapeutics designed to promote apoptosis in cells that evade programmed cell death through overexpression of anti-apoptotic BCL-2 family proteins. BCL-2 functions by sequestering pro- apoptotic BH3-only proteins and preventing activation of the mitochondrial apoptotic pathway; thus, its inhibition restores mitochondrial outer membrane permeabilization, cytochrome c release, and downstream caspase activation. Most clinically relevant BCL-2 inhibitors are small-molecule “BH3 mimetics” that bind with high affinity to the hydrophobic groove of BCL- 2, competitively displacing pro-apoptotic factors such as BIM. Venetoclax (ABT-199) is the prototypical and most widely used agent in this class. Other agents include, for example, navitoclax (ABT-263), obatoclax, gossypol.

[0059]

[0051] The dose of a BCL-2 inhibitor, e.g. venetoclax, in a human recipient may be from about 5 to about 2000 mg / day, e.g. at least about 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, and up to about 400 mg, 600 mg, 800 mg, 1000 mg, 1500 mg, 2000 mg or more.

[0060] Immunosuppressive regimens

[0061]

[0052] An anti-TIM-1 conditioning regimen involves the use of monoclonal antibodies targeting T-cell immunoglobulin and mucin domain-containing protein 1 (TIM-1) to modulate the immune system. TIM-1 is expressed on activated T cells and is implicated in T-cell costimulation, immune tolerance, and tissue inflammation. Anti-TIM-1 antibodies can be used to induce immunological tolerance by selectively depleting or modulating TIM-1 -expressing Tcells, particularly alloreactive T cells that contribute to graft rejection or autoimmunity. Anti- TIM-1 antibodies can also skew the immune response toward regulatory T cells (Tregs) and reduce pro-inflammatory cytokine production, promoting a tolerogenic environment. Antibodies of interest for human use include, for example, the antibody portion of CDX-014, i.e. a non-conjugated counterpart. There are several antibodies specific for human TIM-1 include, for example, 1 D12-TIM1 ; mAbcam75295, 3D9F5, and 3A12E10; recombinant rabbit monoclonal antibodies clone 21 H16L1 and clone n208, etc.

[0062]

[0053] The dose of an anti-TIM1 antibody in a human recipient may be from about 1 to about 1000 mg / day, e.g. at least about 1 mg, 5 mg, 10 mg, 20 mg, 50 mg, 100 mg, and up to about 400 mg, 600 mg, 800 mg, 1000 mg or more.

[0063]

[0054] Total lymphoid irradiation (TLI) is a targeted radiation therapy designed to suppress the immune system by irradiating lymphoid tissues, including lymph nodes, thymus, spleen, and portions of the bone marrow, while sparing other organs. It is commonly used as part of conditioning regimens for organ transplantation, hematopoietic stem cell transplantation, or to treat autoimmune diseases. TLI works by depleting T and B lymphocytes, reducing the risk of graft rejection and graft-versus-host disease (GVHD) while promoting immune tolerance. The treatment typically involves delivering a total dose of 20-40 Gray (Gy), fractionated into smaller doses (e.g., 1.5-2 Gy per session) over several weeks to minimize toxicity to non-lymphoid tissues. The precise dose and schedule depend on the clinical indication and patient-specific factors.

[0064]

[0055] Low-dose total body irradiation (LD-TBI) is a form of radiation therapy in which the entire body is exposed to uniformly distributed, low doses of ionizing radiation. It is commonly used as part of conditioning regimens for hematopoietic stem cell transplantation, particularly in patients with hematologic malignancies, or as a form of immunosuppression for treating autoimmune diseases. LD-TBI primarily functions by depleting immune cells, such as lymphocytes, while sparing other tissues from significant toxicity. The total dose typically ranges from 2 to 6 Gray (Gy), delivered in single or fractionated doses (e.g., 0.2-0.3 Gy per session) over one or more days, depending on the clinical context. LD-TBI is advantageous due to its lower toxicity compared to high-dose regimens and its ability to create a less myeloablative yet immunosuppressive environment, making it suitable for patients who may not tolerate more intensive therapies.

[0065]

[0056] “Major histocompatibility complex antigens” (“MHO”, also called “human leukocyte antigens”, HLA) are protein molecules expressed on the surface of cells that confer a unique antigenic identity to these cells. MHC / HLA antigens are target molecules that are recognizedby T-cells and natural killer (NK) cells as being derived from the same source of hematopoietic stem cells as the immune effector cells ("self") or as being derived from another source of hematopoietic reconstituting cells ("non-self"). Two main classes of HLA antigens are recognized: HLA class I and HLA class II. HLA class I antigens (A, B, and C in humans) render each cell recognizable as "self," whereas HLA class II antigens (DR, DP, and DQ in humans) are involved in reactions between lymphocytes and antigen presenting cells. Both have been implicated in the rejection of transplanted organs.

[0066]

[0057] An important aspect of the HLA gene system is its polymorphism. Each gene, MHC class I (A, B and C) and MHC class II (DP, DQ and DR) exists in different alleles. HLA alleles are designated by numbers and subscripts. For example, two unrelated individuals may carry class I HLA-B, genes B5, and Bw41 , respectively. Allelic gene products differ in one or more amino acids in the a and / or p domain(s). Large panels of specific antibodies or nucleic acid reagents are used to type HLA haplotypes of individuals, using leukocytes that express class I and class II molecules. The genes most important for HLA typing are the six MHC Class I and Class II proteins, two alleles for each of HLA-A; HLA-B and HLA-DR.

[0067]

[0058] The HLA genes are clustered in a “super-locus” present on chromosome position 6p21 , which encodes the six classical transplantation HLA genes and at least 132 protein coding genes that have important roles in the regulation of the immune system as well as some other fundamental molecular and cellular processes. The complete locus measures roughly 3.6 Mb, with at least 224 gene loci. One effect of this clustering is that “haplotypes”, i.e. the set of alleles present on a single chromosome, which is inherited from one parent, tend to be inherited as a group. The set of alleles inherited from each parent forms a haplotype, in which some alleles tend to be associated together. Identifying a patient's haplotypes can help predict the probability of finding matching donors and assist in developing a search strategy, because some alleles and haplotypes are more common than others and they are distributed at different frequencies in different racial and ethnic groups.

[0068]

[0059] As used herein, the term “HLA matched” refers to a donor recipient pair in which none of the HLA antigens are mismatched between the donor and recipient. HLA matched (i.e., where all of the 6 alleles are matched) donor / recipient pairs have a decreased risk of graft v. host disease (GVHD) relative to mismatched pairs (i.e. where at least one of the 6 alleles is mismatched). HLA haploidentical refers to a match where one chromosome is matched at least at HLA-A; HLA-B and HLA-DR, and may be matched at minor histocompatibility loci on the chromosome; but is not necessarily matched on the second chromosome. Such donors frequently occur in families, e.g. a parent is haploidentical to a child; and siblings may be haploidentical.

[0060] As used herein, the term “HLA mismatched” refers to a donor recipient pair in which at least one HLA antigen, in particular with respect to HLA-A, HLA-B and HLA-DR, is mismatched between the donor and recipient. In some cases, one haplotype is matched and the other is mismatched. This situation is frequently found with organs from living or deceased donors. HLA mismatched donor / recipient pairs have an increased risk of GVHD relative to perfectly matched pairs (i.e. where all 6 alleles are matched).

[0069]

[0061] HLA alleles are typically noted with a variety of levels of detail. Most designations begin with HLA- and the locus name, then * and some (even) number of digits specifying the allele. The first two digits specify a group of alleles. Older typing methodologies often could not completely distinguish alleles and so stopped at this level. The third through fourth digits specify a synonymous allele. Digits five through six denote any synonymous mutations within the coding frame of the gene. The seventh and eighth digits distinguish mutations outside the coding region. Letters such as L, N, Q, or S may follow an allele's designation to specify an expression level or other non-genomic data known about it. Thus, a completely described allele may be up to 9 digits long, not including the HLA-prefix and locus notation.

[0070]

[0062] As used herein, a “recipient” is an individual to whom an organ, tissue or cells from another individual (donor), commonly of the same species, has been transferred. For the purposes of the present disclosure, a recipient and a donor are either HLA-matched or HLA- mismatched.

[0071]

[0063] Fc receptors. The human IgG receptor family consists of a number of high affinity Fc receptors, including hFcyRI, hFcyRIIA, hFcyRIIC, hFcyRIIIA, hFcyRIIB, hFcyRIIIB; and a low affinity receptor, hFcRn, involved in recycling and transport of IgG. Activation of the high affinity receptors may require the FcR subunit to be expressed and functional at the cell surface. Other high affinity Fc receptors include, for example, FcaRI (CD89), Fca / p.R, FceRI, etc.

[0072]

[0064] Expression of the Fc receptors varies among immune effector cells. hFcyRI (CD64) is restricted to monocytes / macrophages and dendritic cells (DCs) and, inducibly, expressed on neutrophils and mast cells; hFcyRIIA (CD32A) is expressed on all myeloid cells but not on lymphocytes; hFcyRIIB (CD32B) is highly expressed only on circulating B cells and basophils and expressed on tissue macrophages and DCs, but not on mast cells; hFcyRI IC (CD32C) is expressed on NK cells, monocytes, and neutrophils; hFcyRI HA (CD16A) is expressed on NK cells and monocytes / macrophages; hFcyRIIIB (CD16B) is expressed on neutrophils and subsets of basophils.

[0065] The low affinity hFcRn, which importantly contributes to the biological half-life of antibodies in the blood, is expressed on antigen-presenting cells, monocytes / macrophages, neutrophils, vascular endothelial cells, intestinal epithelial cells, and syncytiotrophoblasts.

[0073]

[0066] The Fey receptors differ in their affinity for I g G and likewise the different IgG subclasses have unique affinities for each of the Fey receptors. These interactions are further tuned by glycans (oligosaccharide), e.g. at position CH2-84.4 of IgG. For example, by creating steric hindrance, fucose containing CH2-84.4 glycans reduce IgG affinity for FcyRIIIA.

[0074]

[0067] Fc domain or region. The Fc region of an antibody mediates its serum half-life and effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). Engineering the Fc region of a therapeutic monoclonal antibody or Fc fusion protein allows the generation of molecules that are better suited to the pharmacology activity required of them. The half-life of an IgG depends on its pH-dependent binding to the neonatal receptor FcRn. FcRn, which is expressed on the surface of endothelial cells, binds the IgG in a pH-dependent manner and protects it from degradation.

[0075]

[0068] A “wild-type Fc region” possesses the effector functions of a native-sequence Fc region, in particular for the purposes of the present invention interacting with one or more of the high affinity receptors e.g. the FcyRI; FcyRIIA; FcyRIIBI ; FcyRIIB2; FcyRIIIA; FcyRIIIB receptors; and can be assessed using various assays as disclosed, for example, in definitions herein. A “dead” Fc is one that has been mutagenized to retain activity with respect to, for example, prolonging serum half-life, but which has reduced or absent binding to a high affinity Fc receptor, including without limitation a human FcyR.

[0076]

[0069] A “native-sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native-sequence human Fc regions include a native-sequence human lgG1 Fc region (non-A and A allotypes); native-sequence human lgG2 Fc region; native-sequence human lgG3 Fc region; and native-sequence human lgG4 Fc region, as well as naturally occurring variants thereof.

[0077]

[0070] A “variant Fc region” or “engineered Fc region” comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a nativesequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology with a native-sequence Fc region and / orwith an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

[0078]

[0071] Variant Fc sequences for a “dead Fc” may include three amino acid substitutions in the CH2 region to reduce FcyRI binding at EU index positions 234, 235, and 237 (see Duncan et al., (1988) Nature 332:563). Two amino acid substitutions in the complement C1q binding site at EU index positions 330 and 331 reduce complement fixation (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991 )). Substitution into human lgG1 of lgG2 residues at positions 233-236 and lgG4 residues at positions 327, 330 and 331 greatly reduces ADCC and CDC (see, for example, Armour KL. etal., 1999 Eur J Immunol. 29(8):2613-24; and Shields RL. etal., 2001. J Biol Chem. 276(9):6591-604).

[0079]

[0072] Binding of IgG to the FcyRs or C1 q depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for FcyRs and C1q binding, and have unique sequences in lgG2 and lgG4. Substitutions into human lgG1 or lgG2 residues at positions 233-236 and lgG4 residues at positions 327, 330 and 331 have been shown to greatly reduce ADCC and CDC. Numerous mutations have been made in the CH2 domain of human IgG 1.

[0080]

[0073] The triple amino acid substitution L234A, L235A, and G237A largely eliminates FcyR and complement effector functions (see, for example, US20100266505).

[0081]

[0074] In some embodiments the Fc region has been modified by the choice of expression host, enzymatic treatment of amino acid substitutions to have reduced glycosylation and binding to FcyR, relative to the native protein. Mutations that reduce binding to FcyR include, without limitation, modification of the glycosylation on asparagine 297 of the Fc domain, which is known to be required for optimal FcR interaction. For example known amino acid substitutions include N297 mutations, for example N297A / Q / D / H / G / C, which changes result in the loss of a glycosylation site on the protein. Enzymatically deglycosylated Fc domains, recombinantly expressed antibodies in the presence of a glycosylation inhibitor and the expression of Fc domains in bacteria have a similar loss of glycosylation and consequent binding to FcyRs.

[0082]

[0075] The LALA variant, L234A / L235A, also has significantly reduced FcyR binding; as does E233P / L234V / L235A / G236 + A327G / A330S / P331 S. See, for example, Armour et al. (1999) Eur J Immunol. 29(8) :2613-24. The set of mutations: K322A, L234A and L235A are sufficient to almost completely abolish FcyR and C1q binding. A set of three mutations, L234F / L235E / P331 S (dubbed TM), have a very similar effect.

[0083]

[0076] Other Fc variants are possible, including without limitation one in which a region capable of forming a disulfide bond is deleted, or in which certain amino acid residues are eliminated at the N-terminal end of a native Fc form or a methionine residue is added thereto.

[0077] The Fc may be in the form of having native sugar chains, increased sugar chains compared to a native form or decreased sugar chains compared to the native form, or may be in an aglycosylated or deglycosylated form. The increase, decrease, removal or other modification of the sugar chains may be achieved by methods common in the art, such as a chemical method, an enzymatic method or by expressing it in a genetically engineered production cell line. Such cell lines can include microorganisms, e.g. Pichia Pastoris, and mammalians cell line, e.g. CHO cells, that naturally express glycosylating enzymes. Further, microorganisms or cells can be engineered to express glycosylating enzymes, or can be rendered unable to express glycosylation enzymes (See e.g., Hamilton, et al., Science, 313:1441 (2006); Kanda, et al, J. Biotechnology, 130:300 (2007); Kitagawa, et al., J. Biol. Chem., 269 (27): 17872 (1994); Ujita-Lee et al., J. Biol. Chem., 264 (23): 13848 (1989); Imai- Nishiya, et al, BMC Biotechnology 7:84 (2007); and WO 07 / 055916). As one example of a cell engineered to have altered sialy lation activity, the alpha-2, 6-sialyltransferase 1 gene has been engineered into Chinese Hamster Ovary cells and into sf9 cells. Antibodies expressed by these engineered cells are thus sialylated by the exogenous gene product. A further method for obtaining Fc molecules having a modified amount of sugar residues compared to a plurality of native molecules includes separating said plurality of molecules into glycosylated and nonglycosylated fractions, for example, using lectin affinity chromatography (See e.g., WO 07 / 117505). The presence of particular glycosylation moieties has been shown to alter the function of Immunoglobulins. For example, the removal of sugar chains from an Fc molecule results in a sharp decrease in binding affinity to the C1q part of the first complement component C1 and a decrease or loss in antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), thereby not inducing unnecessary immune responses in vivo. Additional important modifications include sialylation and fucosylation: the presence of sialic acid in IgG has been correlated with anti-inflammatory activity (See e.g., Kaneko, et al, Science 313:760 (2006)), whereas removal of fucose from the IgG leads to enhanced ADCC activity (See e.g., Shoj-Hosaka, et al, J. Biochem., 140:777 (2006)).

[0084]

[0078] The term “Fc-region-comprising antibody” refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by recombinant engineering the nucleic acid encoding the antibody. Accordingly, an antibody having an Fc region according to this invention can comprise an antibody with or without K447.

[0085]

[0079] In some embodiments, an anti-CD3 antibody used on the methods of the disclosure has impaired or absent binding to a high affinity Fc receptor.

[0080] As used herein, "antibody" includes reference to an immunoglobulin molecule immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies. The term "antibody" also includes antigen binding forms of antibodies, including fragments with antigenbinding capability (e.g., Fab', F(ab’)2, Fab, Fv and rlgG. The term also refers to recombinant single chain Fv fragments (scFv). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies.

[0086]

[0081] Selection of antibodies may be based on a variety of criteria, including selectivity, affinity, cytotoxicity, efc. The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein sequences at least two times the background and more typically more than 10 to 100 times background.

[0087]

[0082] An antibody immunologically reactive with a particular antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, or by immunizing an animal with the antigen or with DNA encoding the antigen. Methods of preparing polyclonal antibodies are known to the skilled artisan. The antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods. In a hybridoma method, an appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.

[0088]

[0083] Human antibodies can be produced using various techniques known in the art, including phage display libraries. Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.

[0089]

[0084] Antibodies also exist as a number of well-characterized fragments produced by digestion with various peptidases. Thus pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CHI by a disulfide bond. The F(ab)'2may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)’2dimer into anFab' monomer. The Fab' monomer is essentially Fab with part of the hinge region. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries.

[0090]

[0085] A "humanized antibody" is an immunoglobulin molecule which contains minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0091]

[0086] Hematopoietic Stem Cells. Hematopoietic stem cells can be obtained by harvesting from bone marrow or from peripheral blood. Bone marrow is generally aspirated from the posterior iliac crests while the donor is under either regional or general anesthesia. Additional bone marrow can be obtained from the anterior iliac crest. A dose of 1 X 108and 2 X 108marrow mononuclear cells per kilogram is generally considered desirable to establish engraftment in autologous and allogeneic marrow transplants, respectively. Bone marrow can be primed with granulocyte colony-stimulating factor (G-CSF; filgrastim [Neupogen]) to increase the stem cell count. Reference to “whole bone marrow” for the purposes described herein generally refers to a composition of mononuclear cells derived from bone marrow that have not been selected for specific immune cell subsets. “Fractionated bone marrow” may be, for example, depleted of T cells, e.g. CD8+cells, CD52+cells, CD3+cells, etc.; enriched for CD34+ cells, etc.

[0092]

[0087] Hematopoietic stem cells are also obtained from cord blood. Cord blood is an almost unlimited source of hematopoietic stem cells for allogeneic hematopoietic stem cell transplant.Cord blood banks (CBB) have been established for related or unrelated UCBT with more than 400,000 units available and more than 20,000 umbilical cord blood transplants performed in children and in adults. UCB hematopoietic progenitors are enriched in primitive stem / progenitor cells able to produce in vivo long-term repopulating stem cells. However, the number of cells available from any single donor can be relatively low in comparison with other sources.

[0093]

[0088] Mobilization of stem cells from the bone marrow into peripheral blood by cytokines such as G-CSF or GM-CSF has led to the widespread adoption of peripheral blood progenitor cell collection by apheresis for hematopoietic stem cell transplantation. The dose of G-CSF used for mobilization is 10 pg / kg / day. In autologous donors who are heavily pretreated, however, doses of up to 40 ,ug / kg / day can be given. Mozobil may be used In conjunction with G-CSF to mobilize hematopoietic stem cells to peripheral blood for collection.

[0094]

[0089] The dose of stem cells administered may depend on the desired purity of the infused cell composition, and the source of the cells. Current guidelines indicate that the minimum dose required for engraftment is 1-2 x 10sCD34+cells / kg body weight for autologous and allogeneic transplants. Higher doses can include, for example, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 107or more. Frequently the dose is limited by the number of available cells. Typically, regardless of the source, the dose is calculated by the number of CD34+ cells present. The percent number of CD34+cells can be low for unfractionated bone marrow or mobilized peripheral blood; in which case the total number of cells administered is much higher.

[0095]

[0090] The CD34+ cells may be selected by affinity methods, including without limitation magnetic bead selection, flow cytometry, and the like from the donor hematopoietic cell sample. The HSPC composition may be at least about 50% pure, as defined by the percentage of cells that are CD34+ in the population, may be at least about 75% pure, at least about 85% pure, at least about 95% pure, or more. Preferable a maximum number of CD3+ cells delivered with the HSPC composition is not more than about 106CD3+cells / kg of recipient body weight, not more than about 105CD3+cells / kg of recipient body weight, not more than about 104CD3+cells / kg of recipient body weight. Alternatively cell populations may be tandemly selected for expression of CD34 and CD90, which cell populations may be highly purified, e.g. at least about 85% CD34+CD90+cells, at least about 90% CD34+CD90+cells, at least about 95% CD34+CD90+cells and may be up to about 99% CD34+CD90+cells or more. Alternatively unmanipulated bone marrow or mobilized peripheral blood populations are used.

[0096]

[0091] Hematopoietic stem cells can also be generated in vitro, for example from pluripotent embryonic stem cells, induced pluripotent cells, and the like. For example, see Sugimura etal. (2017) Nature 545:432-438, herein specifically incorporated by reference, which details a protocol for generation of hematopoietic progenitors.

[0097]

[0092] The cells which are employed may be fresh, frozen, or have been subject to prior culture. They may be fetal, neonate, adult, etc. Hematopoietic stem cells may be obtained from fetal liver, bone marrow, blood, particularly G-CSF or GM-CSF mobilized peripheral blood, or any other conventional source. Cells for engraftment are optionally isolated from other cells, where the manner in which the stem cells are separated from other cells of the hematopoietic or other lineage is not critical to this invention. If desired, a substantially homogeneous population of stem or progenitor cells may be obtained by selective isolation of cells free of markers associated with differentiated cells, while displaying epitopic characteristics associated with the stem cells.

[0098]

[0093] Chimerism, as used herein, generally refers to chimerism of the hematopoietic system, unless otherwise noted. A determination of whether an individual is a full chimera, mixed chimera, or non-chimeric made be made by an analysis of a hematopoietic cell sample from the graft recipient, e.g. peripheral blood, bone marrow, etc. as known in the art. Analysis may be done by any convenient method of typing. In some embodiments the degree of chimerism amongst all mononuclear cells, T cells, B cells, CD56+ NK cells, and CD15+ neutrophils is regularly monitored, using PCR with probes for microsatellite analysis. For example, commercial kits that distinguish polymorphisms in short terminal repeat lengths of donor and host origin are available. Automated readers provide the percentage of donor type cells based on standard curves from artificial donor and host cell mixtures.

[0099]

[0094] Individuals who exhibited more than a 95% donor cells in a given blood cell lineage by such analysis at any time post-transplantation are referred to as having full donor chimerism in this transplant patient group. Mixed chimerism is defined as greater than 1 % donor but less than 95% donor DNA in such analysis. Individuals who exhibit mixed chimerism may be further classified according to the evolution of chimerism, where improving mixed chimerism is defined as a continuous increase in the proportion of donor cells over at least a 6-month period. Stable mixed chimerism is defined as fluctuations in the percentage of recipient cells over time, without complete loss of donor cells.

[0100]

[0095] A determination of whether a subject is a full chimera, mixed chimera, or non-chimera can be made by an analysis of a hematopoietic cell sample from the graft recipient, e.g. peripheral blood or bone marrow. Analysis can be done by any convenient method of typing. In some embodiments, the degree of chimerism amongst all mononuclear cells, T cells, B cells, CD56+ NK cells, and CD15+ neutrophils is regularly monitored, using PCR with probes for microsatellite analysis. For example, commercial kits can be used to quantify donor andhost genetic material extracted from cells based on polymorphisms in short terminal repeat lengths. Automated readers provide the percentage of donor type cells based on standard curves from artificial donor and host cell mixtures.

[0101]

[0096] A "patient" for the purposes of the present invention includes both humans and other animals, particularly mammals, including pet and laboratory animals, e.g. mice, rats, rabbits, etc. Thus the methods are applicable to both human therapy and veterinary applications. In one embodiment the patient is a mammal, preferably a primate. In other embodiments the patient is human.

[0102]

[0097] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and / or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and / or symptom(s), i.e. , arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and / or symptom(s). Those in need of treatment include those already inflicted (e.g., those with cancer, those with an infection, etc.) as well as those in which prevention is desired (e.g., those with increased susceptibility to cancer, those with an increased likelihood of infection, those suspected of having cancer, those suspected of harboring an infection, etc.).

[0103] Methods of Use

[0104]

[0098] Compositions and methods are provided for improved T cell lymphodepletion in a mammalian host, using a conditioning regimen wherein concomitant treatment of an effective dose of an antibody specific for CD3 and an effective dose of a BCL2 inhibitor provides for improved T cell depletion. The treatment induces T cell apoptosis, which can trigger changes that improve immune tolerance, including an increase in the number of one or more of regulatory (Treg) cells, myeloid derived suppressor cells (MDSC), and tolerogenic dendritic cells (DC2). The regimen finds use, for example, in conditioning a recipient for transplantation of hematopoietic stem cells (HSCT), for solid organ transplantation, for transplantation of chimeric antigen receptor T cells (CAR-T), for treatment or prevention of graft versus host disease (GVHD), and the like.

[0099] In some embodiments the conditioning regimen is combined with an immunosuppressive regimen. In some embodiments the immunosuppressive regimen comprises administration of an effective dose of an antibody specific for TIM1. In some embodiments the immunosuppressive regimen comprises administration of total lymphoid irradiation. In some embodiments the immunosuppressive regimen comprises administration of a low dose total body irradiation. In some embodiments the administration is performed in the absence of anti-thymocyte globulin (ATG). In some embodiments the administration is performed in the absence of cyclophosphamide.

[0105] [ o] In some embodiments the antibody specific for CD3 specifically binds to human CD3.

[0106] In some embodiments the antibody specific for CD3 lacks an Fc region sequence. In some embodiments the antibody specific for CD3 comprises an Fc sequence modified to reduce binding to high affinity FcyR. In some embodiments the antibody specific for CD3 is an F(ab)2 fragment, or an F(ab’)2 fragment. In some embodiments the anti-CD3 antibody is a fragment or derivative of muromonab-CD3, otelixizumab, teplizumab, foralumab or visilizumab.

[0107]

[0101] BCL-2 inhibitors target the anti-apoptotic protein B-cell lymphoma 2 (BCL-2), which plays a critical role in cell survival by inhibiting apoptosis. In some embodiments the BCL2 inhibitor is venetoclax (ABT-199), a selective small-molecule inhibitor approved for treating chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). In other embodiments the BCL2 inhibitor is navitoclax (ABT-263); S55746 (BCL201); Obatoclax (GX15-070); gossypol, etc.

[0108]

[0102] In some embodiments the conditioning regimen of the disclosure is administered prior to bone marrow transplantation. In some embodiments the conditioning regimen of the disclosure is administered prior to hematopoietic stem cell transplantation. In some embodiments the conditioning regimen of the disclosure is administered prior to CAR-T cell administration. In some embodiments the conditioning regimen of the disclosure is administered prior to solid organ transplantation, where the solid organ transplantation is optionally combined with BMT or HSCT. In some embodiments the conditioning regimen of the disclosure is administered for the treatment of GVHD, where administration may be prior to, or following transplantation of hematopoietic cells. In some such embodiments administration follows a determination of GVHD. In some embodiments the conditioning regimen of the disclosure is administered for the treatment of an autoimmune disease. The methods allow engraftment to treat hematologic disorders, to reduce undesirable T cell reactivity and to tolerize a recipient to a donor-type HLA for organ transplantation.

[0109]

[0103] The dose of each agent of interest can be administered, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth. Generally the conditioning agents are administered 1 , 2, 3, 4 or more days prior to and consequent tohematopoietic cell administration. For example, in some embodiments, an antibody is administered once every two to four days for a period of a week to 10 days. By "twice-weekly" or "two times per week" is intended that two therapeutically effective doses of the agent in question is administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By "thrice weekly" or "three times per week" is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses.

[0110]

[0104] The therapeutic dose of an agent may be at least about 0.01 pg / kg body weight, at least about 0.05 pg / kg body weight; at least about 0.1 pg / kg body weight, at least about 0.5 pg / kg body weight, at least about 1 pg / kg body weight, at least about 2.5 pg / kg body weight, at least about 5 pg / kg body weight, etc. It will be understood by one of skill in the art that such guidelines will be adjusted for the identity of the active agent. The agents can be administered by any acceptable route of administration as noted herein below, e.g. intranasal, inhalation, etc., or for systemic administration, e.g. i.m., i.p., i.v., and the like, usually parental administration for antibodies. Venetoclax may be orally administered.

[0111]

[0105] Hematopoietic stem cell transplantation (HCT) is the transplantation of multipotent hematopoietic stem and progenitor cells (HSPCs), usually derived from bone marrow, peripheral blood, or umbilical cord blood. HSPCs can have extensive self-renewal capacity, and an ability to differentiate into specialized cell types, for example, an ability to reconstitute all hematopoietic cell lineages. HSPCs can undergo asynchronous replication, where two daughter cells are produced with different phenotypes. Hematopoietic stem cells can exist in a mitotically quiescent form.

[0112]

[0106] HSPCs can be obtained by harvesting from bone marrow or from peripheral blood.

[0113] Bone marrow can be aspirated from the posterior iliac crest or the anterior iliac crest while the donor is under either regional or general anesthesia. HSPCs can be obtained by harvesting from peripheral blood, for example, by peripheral blood apheresis. The number of stem cells harvested can be increased by treating the donor with a mobilization agent, i.e. an agent that mobilizes stem cells from the bone marrow into peripheral blood. Non-limiting examples of mobilization agents include granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), mozobil, and combinations thereof. T echniques to mobilize stem cells into peripheral blood can comprise administering to a donor, for example, 10 to 40 p / kg / day of a mobilization agent. A mobilization agent can be administered to the donor in, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses. An apheresis product can be isolated from a donor about, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12,13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 26, 28, or 30 hour(s) after a dose of mobilization agent.

[0114]

[0107] The methods of the disclosure can be used for treating a human subject with a cancer.

[0115] In some embodiments, the subject has been treated for cancer, e.g. by treatment with a chemotherapeutic drug or with radiation. The methods of the disclosure can be useful for treating, a hematologic malignancy, for example, leukemia or lymphoma. Examples of hematologic malignancies that can be treated by the methods of the disclosure include, but are not limited to, acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), multiple myeloma, and lymphomas such as Hodgkin and non-Hodgkin lymphomas. A cancer can be a solid tumor. In some embodiments, the cancer is a primary or metastatic tumor.

[0116]

[0108] The types of cancer that can be treated using the methods of the present invention include but are not limited to leukemia, lymphoma, adrenal cortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain cancers, central nervous system (CNS) cancers, peripheral nervous system (PNS) cancers, breast cancer, cervical cancer, childhood Non-Hodgkin's lymphoma, colon and rectum cancer, endometrial cancer, esophagus cancer, Ewing's family of tumors (e.g. Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, hairy cell leukemia, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and pharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, children's leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, liver cancer, lung cancer, lung carcinoid tumors, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, melanoma skin cancer, nonmelanoma skin cancers, stomach cancer, testicular cancer; thymus cancer, thyroid cancer, uterine cancer (e.g. uterine sarcoma), transitional cell carcinoma, vaginal cancer, vulvar cancer, mesothelioma, squamous cell or epidermoid carcinoma, bronchial adenoma, choriocarinoma, head and neck cancers, teratocarcinoma, and Waldenstrom's macroglobulinemia.

[0117] Hematopoietic Cell Transplantation and Graft Versus Host Disease

[0118]

[0109] The present disclosure provides methods for improved hematopoietic stem cell transplantation (HCT), wherein GVHD is reduced or prevented. In some embodiments, an HCT recipient subject is administered treatment described herein, and the recipient subjectdoes not develop GVHD within certain spans of time after administration (e.g, within 30, 100, or 200 days). For example, the likelihood of the recipient subject developing GVHD can be reduced within certain spans of time after administration, relative to a subject receiving HCT according to a different protocol (e.g, within 30, 100, or 200 days).

[0119]

[0110] Graft-versus-host disease (GVHD) is an inflammatory disease that can occur in the allogenic transplant setting. GVHD involves donor cells (graft) attacking recipient cells (host). GVHD can be classified into acute GVHD (aGVHD) and chronic GVHD (cGVHD). aGVHD typically occurs in the first 3 months after transplantation. aGVHD can be life-threatening and can involve, for example, the skin, the intestines, and / or the liver. cGVHD typically occurs after the first 3 months following transplant. cGVHD is a major source of late treatment-related complications, and can be life-threatening. In addition to inflammation, cGVHD can lead to the development of fibrosis, which can result in functional disability.

[0120]

[0111] The early morbidity and mortality associated with acute graft versus host disease (aGVHD) is a major factor limiting the success of HCT, as is the long-term morbidity associated with chronic GVHD (cGVHD). The incidence of aGVHD following allogeneic HCT from an HLA- matched sibling donor (MSD) is 20 to 60%, despite the use of various immunosuppressive agents such as tacrolimus, cyclosporine, methotrexate, mycophenolate, anti-thymocyte globulin and corticosteroids. Approximately one-third of patients who undergo allogeneic HCT using a MSD and a T cell replete graft will develop chronic GVHD.

[0121]

[0112] GVHD severity can be graded, for example, using the Glucksberg grade (l-IV) or the International Bone Marrow Transplant Registry (IBMTR) grading system (A-D). The severity of acute GVHD is determined by an assessment of the degree of involvement of the skin, liver, and gastrointestinal tract. The stages of individual organ involvement are combined with (Glucksberg) or without (IBMTR) the patient’s performance status to produce an overall grade, which can have prognostic significance. Grading is important in terms of assessing the response to prophylaxis or treatment, impact upon survival, and association with graft-versus- leukemia effect.

[0122]

[0113] Grade l(A) GVHD is characterized as mild disease, grade ll(B) GVHD as moderate, grade lll(C) as severe, and grade IV(D) life-threatening. The IBMTR grading system defines the severity of acute GVHD as follows: Grade A: stage 1 skin involvement alone (maculopapular rash over <25 percent of the body) with no liver or gastrointestinal involvement; Grade B: stage 2 skin involvement, stage 1 to 2 gut or liver involvement; Grade C: stage 3 involvement of any organ system (generalized erythroderma; bilirubin 6.1 to 15.0 mg / dL; diarrhea 1500 to 2000 mL / day); Grade D: stage 4 involvement of any organ system (generalized erythroderma with bullous formation; bilirubin >15 mg / dL; diarrhea >2000 mL / day OR pain OR ileus). Patients with moderate to severe GVHD have a significantly highermortality rate compared with those with milder disease, for example, estimated five year survival for patients with grade III (C) aGVHD is 25%, while for patients with grade IV (D) estimated five year survival is 5%.

[0123]

[0114] Management of GVHD may require immunosuppressive therapy (for example, high dose corticosteroids, prolonged administration of immunosuppressants) or cytotoxic mediations, all of which are associated with toxicity. In many cases, immunosuppressive therapies can fail to effectively treat GVHD, or can result in increased susceptibility to infection, or blunted anti-tumor immunity.

[0124]

[0115] In some embodiments, the methods disclosed herein prevent or reduce GVHD in an HCT recipient subject. For example, the methods disclosed herein can prevent any manifestation of GVHD in a subject receiving HCT.

[0125]

[0116] The methods disclosed herein can prevent, for example, any GVHD of stage 1 or above, any GVHD of stage 2 or above, any GVHD of stage 3 or above, or any GVHD of stage 4 in subjects receiving HCT.

[0126] Solid Organ Transplant

[0127]

[0117] Solid organs may be transplanted from a donor to a recipient such that the organ is placed into the appropriate position in the recipient body. In some cases, the cardiovascular connections between the solid organ may be physiologically integrated into the recipient body. In some cases, the organ may be from a living donor. In other cases, the organ may be from a deceased donor. In some cases, the solid organ may be HLA-matched between the donor and the recipient. In other cases, the solid organ may be HLA-mismatched between the donor and the recipient.

[0128]

[0118] Any solid organ that may be used for organ transplantation may be used with the methods described herein. In some cases, the organ may be a kidney, lung, pancreas, pancreatic islet cells, heart, intestine, colon, liver, skin, muscle, gum, eye, tooth and the like as known to those of skill in the art. In some cases, the organ may be a complete organ. In other cases, the organ may be a portion of an organ. In other cases, the organ may be cells from a tissue of an organ.

[0129]

[0119] Using the methods described herein, the solid organ is harvested and transplanted in accordance with conventional practice following the conditioning regimen of the disclosure.

[0130] Autoimmune disease

[0131]

[0120] In yet other embodiments, the conditioning regimen is used for the treatment of autoimmune disease. The spectrum of inflammatory diseases and diseases associated with inflammation is broad and includes autoimmune diseases such rheumatoid arthritis (RA),systemic lupus erythematosus (SLE), multiple sclerosis (MS), and autoimmune hepatitis; insulin dependent diabetes mellitus, degenerative diseases such as osteoarthritis (OA), Alzheimer’s disease (AD), and macular degeneration.

[0132]

[0121] Many, if not most, autoimmune and inflammatory diseases involve multiple types of T cells, e.g. TH1 , TH2, TH17, and the like. Autoimmune diseases are characterized by T and B lymphocytes that aberrantly target self-proteins, -polypeptides, -peptides, and / or other selfmolecules causing injury and or malfunction of an organ, tissue, or cell-type within the body (for example, pancreas, brain, thyroid or gastrointestinal tract) to cause the clinical manifestations of the disease. Autoimmune diseases include diseases that affect specific tissues as well as diseases that can affect multiple tissues, which can depend, in part on whether the responses are directed to an antigen confined to a particular tissue or to an antigen that is widely distributed in the body.

[0133]

[0122] The indications for treatment vary according to disease categories and are influenced by factors such as cancer type, cytogenetic abnormalities, response to prior therapy, patient age and performance status, disease status (remission vs relapse), disease-specific prognostic factors, availability of a suitable graft source, time of referral, and time to transplant.

[0134]

[0123] Autologous HSCT is currently used to treat the following conditions: Multiple myeloma, Non-Hodgkin lymphoma, Hodgkin disease, Acute myeloid leukemia, Neuroblastoma, Germ cell tumors, Autoimmune disorders - Systemic lupus erythematosus (SLE), systemic sclerosis, Amyloidosis.

[0135]

[0124] Allogenic HSCT is currently used to treat the following disorders: Acute myeloid leukemia, Acute lymphoblastic leukemia, Chronic myeloid leukemia; Chronic lymphocytic leukemia, Myeloproliferative disorders, Myelodysplastic syndromes, Multiple myeloma, NonHodgkin lymphoma, Hodgkin disease, Aplastic anemia, Pure red cell aplasia, Paroxysmal nocturnal hemoglobinuria, Fanconi anemia, Thalassemia major, Sickle cell anemia, Severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, Hemophagocytic lymphohistiocytosis (HLH), Inborn errors of metabolism - Eg, mucopolysaccharidosis,, Gaucher disease, metachromatic leukodystrophies, and adrenoleukodystrophies, Epidermolysis bullosa, Severe congenital neutropenia, Shwachman- Diamond syndrome, Diamond-Blackfan anemia, Leukocyte adhesion deficiency, and the like.

[0136]

[0125] Embodiments of the invention include transplantation into a patient suffering from a genetic blood disorder, where exogenous stem cells of a normal phenotype are transplanted into the patient. Such diseases include, without limitation, the treatment of anemias caused by defective hemoglobin synthesis (hemoglobinopathies). The stem cells may be allogeneic stem cells of a normal phenotype, or may be autologous cells that have been geneticallyengineered to delete undesirable genetic sequences, and / or to introduce genetic sequences that correct the genetic defect.

[0137]

[0126] Sickle cell diseases include HbS Disease; drepanocytic anemia; meniscocytosis.

[0138] Chronic hemolytic anemia occurring almost exclusively in blacks and characterized by sickleshaped RBCs caused by homozygous inheritance of Hb S. Homozygotes have sickle cell anemia; heterozygotes are not anemic, but the sickling trait (sicklemia) can be demonstrated in vitro. In Hb S, valine is substituted for glutamic acid in the sixth amino acid of the beta chain. Deoxy-Hb S is much less soluble than deoxy-Hb A; it forms a semisolid gel of rodlike tactoids that cause RBCs to sickle at sites of low PO2. Distorted, inflexible RBCs adhere to vascular endothelium and plug small arterioles and capillaries, which leads to occlusion and infarction. Because sickled RBCs are too fragile to withstand the mechanical trauma of circulation, hemolysis occurs after they enter the circulation. In homozygotes, clinical manifestations are caused by anemia and vaso-occlusive events resulting in tissue ischemia and infarction. Growth and development are impaired, and susceptibility to infection increases. Anemia is usually severe but varies highly among patients. Anemia may be exacerbated in children by acute sequestration of sickled cells in the spleen.

[0139]

[0127] Thalassemias are a group of chronic, inherited, microcytic anemias characterized by defective Hb synthesis and ineffective erythropoiesis, particularly common in persons of Mediterranean, African, and Southeast Asian ancestry. Thalassemia is among the most common inherited hemolytic disorders. It results from unbalanced Hb synthesis caused by decreased production of at least one globin polypeptide chain ( , a, y, 8).

[0140]

[0128] Aplastic anemia results from a loss of RBC precursors, either from a defect in stem cell pool or an injury to the microenvironment that supports the marrow, and often with borderline high MOV values. The term aplastic anemia commonly implies a panhypoplasia of the marrow with associated leukopenia and thrombocytopenia.

[0141]

[0129] Combined immunodeficiency is a group of disorders characterized by congenital and usually hereditary deficiency of both B- and T-cell systems, lymphoid aplasia, and thymic dysplasia. The combined immunodeficiencies include severe combined immunodeficiency, Swiss agammaglobulinemia, combined immunodeficiency with adenosine deaminase or nucleoside phosphorylase deficiency, and combined immunodeficiency with immunoglobulins (Nezelof syndrome). Most patients have an early onset of infection with thrush, pneumonia, and diarrhea. If left untreated, most die before age 2. Most patients have profound deficiency of B cells and immunoglobulin. The following are characteristic: lymphopenia, low or absent T-cell levels, poor proliferative response to mitogens, cutaneous anergy, an absent thymic shadow, and diminished lymphoid tissue. Pneumocystis pneumonia and other opportunistic infections are common.EXPERIMENTAL

[0142] Example 1

[0143] Combining a BCL2 inhibitor with anti-CD3 enhances Lymphodepletion

[0144]

[0130] Venetoclax combined with «CD3 F(ab’)2 treatment leads to increased apoptosis of T cells in vitro, lymphodepletion and induction of regulatory T cells and tolerogenic cell subsets in vivo, when combined with anti-TI M 1 conditioning. T cell lymphodepletion, especially of CD8+T cells was induced post Venetoclax, ocCD3 F(ab’)2 and a-TIM-1 conditioning in the spleen, nodes and bone marrow. Regulatory T cells as well as tolerogenic dendritic cells (DC2) were also induced in the spleen and lymph nodes. Similar results were observed when Venetoclax, and aCD3 F(ab’)2 conditioning was performed in combination with Total Lymphoid Irradiation (TLI). Further, this regimen provided for persistent mixed chimerism after bone marrow transplantation (BMT). The combination with low dose total body irradiation (TBI) also leads to lymphodepletion, increased levels of immunoregulatory subsets, including regulatory T cells, tolerogenic CD8 SIRPa+dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) and persistent mixed chimerism after BMT.

[0145]

[0131] When combined with allogeneic islet transplantation, Venetoclax, and aCD3 F(ab’)2 conditioning in combination with low dose total body irradiation (TBI) leads to tolerance to islet allograft. The combination can also be administered post islet transplantation to protect the allogeneic islet graft and alleviate graft versus host disease (GVHD) in the case of induction of full donor chimerism.

[0146]

[0132] Venetoclax, and aCD3 F(ab’)2 treatment post allogeneic BMT protects against GVHD through the induction of regulatory T cells. Venetoclax, and aCD3 F(ab’)2 treatment post BMT is less inflammatory compared to post cyclophosphamide treatment (PTCy). In contrast, PTCy treatment post islet transplantation leads to loss of islet allograft.

[0147] MATERIALS AND METHODS

[0148]

[0133] Mice. Female mice between 8 and 12 wk old were used for the experiments. BALB / c (H-2d, CD45.2), C57BL / 6 (B6) (H-2b, CD45.2), and C57BL / 6 (B6) (H-2b, CD45.1), were purchased from The Jackson Laboratory (Sacramento, GA). C57BL / 6 albino FoxP3 mutant mice expressing diphtheria toxin (DT) receptor, GFP, and luc (FoxP3DTR / GFP / luc) were a kind gift from Dr Gunter J. Hammerling (Heidelberg, Germany) and bred in our animal facility. Animal protocols were approved by the Institutional Animal Care and Use Committee of Stanford University.

[0149]

[0134] Cell isolation. CD4 / CD8 conventional T cells (Tcon), were prepared as described in

[0150] [1].

[0135] Monoclonal antibody, Venetoclax and Cyclophosphamide treatment. Anti-mouse TIM- 1 clone 3D10 (antagonist that blocks PtdSer binding) [2, 3], have been previously described. Mice were injected intraperitoneally (i.p.) with 400 |jg / mouse on days -1 , post allogeneic T cell transplantation. aCD3 F(ab’)2 (BioXcell) was injected i.v for 5 consecutive doses (50 pg / mouse) on day -6 to day -2, aCD3 mAB (BioXcell) was injected on day -5 (50 pg / mouse) and Venetoclax (Chemietek) was administered via oral gavage (p.o) on day-3 and day -2 post allogeneic bone marrow cell transplantation. Venetoclax and Cyclophosphamide (Sigma, 50mg / kg, p.o) was also administered on day+3 and day+4 post allogeneic bone marrow cell transplantation.

[0151]

[0136] Allogeneic bone marrow transplantation. Allogeneic bone marrow transplantation performed as described [1]. Briefly, animals received either total body irradiation (TBI) (250- 500cGy) or 10 doses of total lymphoid irradiation (TLI) combined with 5 doses of antithymocyte globulin (ATS) and 30-50x106allogeneic bone marrow cells.

[0152]

[0137] Islet transplantation model. Islet transplantation was performed as described as described..

[0153]

[0138] Bioluminescence imaging (BLI). BLI was performed as described [5] with an I VIS Spectrum imaging system (Xenogen). Images were analyzed with Living Image Software 4.2 (Xenogen).

[0154]

[0139] Flow cytometry. Data were collected using a BD LSR2 flow cytometer (BD Biosciences) and data were analyzed using FlowJo 10.0.7 software (Tree, Star, Ashland, OR)

[0155] 1. Schneidawind, D., et al., CD4+ invariant natural killer T cells protect from murine GVHD lethality through expansion of donor CD4+CD25+FoxP3+ regulatory T cells. Blood, 2014. 124(22): p. 3320-8.

[0156] 2. Zhou, Z., et al., Neogenin regulation of BMP-induced canonical Smad signaling and endochondral bone formation. Dev Cell, 2010. 19(1): p. 90-102.

[0157] 3. I liopoulou, B.P., et al., Blockade of TIM-1 on the donor graft ameliorates graft-versus- host disease following hematopoietic cell transplantation. Blood Adv, 2019. 3(21): p. 3419- 3431.

[0158] 4. Jeong, J.H., et al., Functional enhancement of beta cells in transplanted pancreatic islets by secretion signal peptide-linked exendin-4 gene transduction. J Control Release, 2012. 159(3): p. 368-75.

[0159] 5. Edinger, M., et al., Revealing lymphoma growth and the efficacy of immune cell therapies using in vivo bioluminescence imaging. Blood, 2003. 101(2): p. 640-8.

[0160] Example 2

[0140] T cell-based immunotherapies are effective for treating hematological malignancies and promoting lifelong graft tolerance after solid organ transplantation. Recipient preconditioning resulting in profound lymphodepletion is a key to preventing graft rejection and maximizing T cell engraftment. The 2 standard lymphodepleting agents, antithymocyte globulin and cyclophosphamide chemotherapy, are associated with major adverse effects in patients. This research investigates a new therapeutic approach that combines anti-CD3 (aCD3) therapy with venetoclax, as a BCL-2 inhibitor, and may be further combined with T cell immunoglobulin and mucin 1 (TIM1) blockade to achieve optimal T cell lymphodepletion and immune tolerance in preclinical hematopoietic stem cell transplantation models. This conditioning regimen strongly induces T cell apoptosis, particularly in naive and central memory CD8+ T cells, and simultaneously enhances regulatory T cells and tolerogenic dendritic cells. This combination therapy improved donor engraftment, promoted allograft tolerance, and prevented graft-versus-host disease in murine hematopoietic stem cell transplantation and pancreatic islet transplantation models. This aCD3 targeted approach is a more precise and less toxic lymphodepletion strategy, as demonstrated by reduced organ damage and inflammatory response, compared to posttransplant cyclophosphamide. Our study introduces a novel aCD3-based conditioning approach which enables successful engraftment, mixed chimerism, and tolerance induction.

[0161]

[0141] Depletion of T cells with anti-CD3 (aCD3) monoclonal antibodies (m Abs) has emerged as a promising strategy because these lymphodepleting agents are specific and have a shorter half-life compared to ATG. Newer aCD3, like teplizumab, are well tolerated and are Food and Drug Administration (FDA) approved for the treatment of stage 2 type 1 diabetes (T1 D). Teplizumab has an inert Fc domain and induces T cell depletion that partially agonizes CD3 and induces programmed cell death in target T cells.

[0162]

[0142] The preclinical murine equivalent commonly used is a F(ab’)2 fragment of the antibody 2C11. Both antibodies induce tolerance signals secondary to apoptotic bodies absorbed by antigen-presenting cells. Additionally, FOXP3+ regulatory T cells (Tregs) can also be resistant to aCD3 treatment. However, autoreactive cells and memory T cells can also be resistant to aCD3 due to higher expression of BCL2, preventing apoptosis. We reasoned that BCL-2 inhibition could synergize with aCD3 to induce deeper T cell depletion and stronger tolerance signals. Venetoclax (ABT-199) is a selective small-molecule BCL2 inhibitor approved for treating hematologic malignancies.

[0163]

[0143] The T cell immunoglobulin and mucin 1 (TIM-1) receptor shows a strong proinflammatory signal when it binds to phosphatidylserine (PtdSer) exposed on the cell surface of cells undergoing apoptosis. Upon binding, there is a potent inflammatory responseon multiple cell types, including invariant natural killer T cells, dendritic cells (DCs), and T cells, while also destabilizing Tregs and regulatory B cells. TIM-1 blockade increases Treg suppressive capacity, promotes tolerance in solid organ transplantation and protects against GVHD in preclinical murine models.

[0164]

[0144] We hypothesized that the combination of aCD3, venetoclax, and immunosuppression, such as TIM-1 blockade creates strong synergy in promoting immune tolerance in tissue and organ transplantation. We observed that this combination improved engraftment and prevented GVHD. When incorporating this approach in either HSCT or HSCT plus islet transplantation, it facilitated the establishment of hematopoietic mixed chimerism and allograft tolerance. This aCD3 targeted approach has the advantage of a more precise and less toxic lymphodepletion strategy better positioned to advance engineered donor grafts and T cellbased therapy products tolerance.

[0165] Materials and methods

[0166]

[0145] Mice. Female mice aged between 8 and 12 weeks were used for the experiments.

[0167] BALB / c (H-2d, CD45.2), C57BL / 6 (B6) (H-2b, CD45.2), and C57BL / 6 (B6) (H-2b, CD45.1) were purchased from The Jackson Laboratory (Sacramento, CA). C57BL / 6 al-bino FoxP3 mutant mice expressing diphtheria toxin receptor (DTR), green fluorescent protein (GFP), and luc (FOXP3DTR / GFP / luc) were a kind gift from Dr Gunter J. Ha€mmerling (Heidelberg, Germany). Animal protocols were approved by the Institutional Animal Care and Use Committee of Stanford University.

[0168]

[0146] Monoclonal antibody, venetoclax, and cyclophosphamide treatment. Antimouse TIM-1 clone 3D10 (antagonist that blocks PtdSer binding) has been previously described.

[0169]

[0147] Bioluminescence imaging. Bioluminescence imaging (BLI) was performed as described with an MS Spectrum imaging system (Xenogen). Images were analyzed with Living Image Software 4.2 (Xenogen).

[0170]

[0148] Flow cytometry. Data were collected using a BD LSR2 flow cytometer (BD Biosciences), and data were analyzed using FlowJo 10.10.0 software (Tree Star, Ashland, OR).

[0171]

[0149] T Cell isolation. CD4 / CD8 conventional T cells (Tcon), were prepared as previously described. Briefly, spleens were collected from age and sex-matched mice and splenocytes suspensions were obtained using a 70um strainer. Isolation of T cells was performed using a T cell isolation kit (EasySep™ Mouse T cell Isolation kit, STEMCELL technologies,) as per protocol’s instructions.

[0172]

[0150] Monoclonal antibody, venetoclax and cyclophosphamide treatment. Anti-mouse TIM- 1 clone 3D10 (antagonist that blocks PtdSer binding), has been previously described. Micewere injected intraperitoneally (i.p.) with 400 pg / mouse TIM-1 mAb on day-4 and day-1 , post allogeneic T cell transplantation. aCD3 F(ab)2(BioXcell) was injected i.v for 5 consecutive doses (50 pg / mouse) on day -6 to day -2, aCD3 mAb (BioXcell) was injected on day -5 (50 pg / mouse) and venetoclax (Chemietek) was administered via oral gavage (p.o) on day-3 and day -2 post allogeneic bone marrow (BM) cell transplantation. Venetoclax and cyclophosphamide (Sigma, 50mg / kg, i.p) were also administered on day +3 and day +4 post allogeneic BM cell transplantation.

[0173]

[0151] Allogeneic bone marrow transplantation. Mice received either total body irradiation (TBI) (250cGy) or 10 doses of total lymphoid irradiation (TLI) combined with 5 doses of antithymocyte globulin (ATS) and 30-50x106allogeneic BM cells. For bone marrow isolation, bone marrow suspensions were prepared from the femur and tibia by flushing the bones with RPMI 1640 containing 10% fetal bovine serum. Cell suspensions were filtered through a 70um strainer and resuspended in sterile phosphate buffered saline. For the MHC mismatched BMT model, mice received BALB / c animals received 5x10sallogeneic T cell depleted (TCD) BM and 10sT cells from MHC major mismatched B6 donors.

[0174]

[0152] Islet transplantation model. Islet transplantation was performed as previously described. Briefly, the kidney was surgically exposed to make a small incision in the renal capsule. Then, 100 luciferase expressing islets were deposited under the capsule of anesthetized mice using a microcapillary tube. The kidney capsule was cauterized, and the incision was closed using sutures and staples. Staples were removed at day 5 post-islet transplantation.

[0175]

[0153] Statistical analysis. Figure legends indicate statistical tests that were used, and P < .05 was considered statistically significant.

[0176] Results

[0177]

[0154] Venetoclax and aCD3 F(ab')2treatment enhances in vitro apoptosis, T cell depletion, and promotes Tregs and tolerogenic cell subsets in vivo. With the recent FDA approval of the aCD3 mAb teplizumab, current clinical depletion of CD3 T cells by antibody targeting could shift from Fc-based lysis to the induction of apoptosis by direct action on the CD3 receptor. Expression of BCL2 by T cells can make them resistant to apoptotic killing. Enhanced T cell depletion may facilitate T cell engraftment without cytokine release syndrome. We, therefore, asked if venetoclax enhances apoptotic cell death initiated by aCD3 F(ab)2(2C11) in vitro (Fig.

[0178] 1 A). Splenocytes were isolated from B6 mice and cultured at rest for 48 hours in the presence of venetoclax (500 nM) and aCD3 F(ab’)2(1 ng / well). Early apoptosis was significantly increased in splenic T cells after treatment with aCD3 F(ab)2and venetoclax compared to treatment with aCD3 F(ab)2alone (Fig. 1 B). Apoptosis can be sensed by various immune receptor pathways, including the cell surface expression of PtdSer. We asked if aCD3 depletionof T cells might be enhanced by TIM-1 blockade by using an aTIM-1 mAb (clone 3D10). We found that the combination of aCD3 F(ab)2, venetoclax, and aTIM-1 mAb did not increase lymphodepletion in vitro, compared to aCD3 F(ab’)2 and venetoclax (Fig. 1B).

[0179]

[0155] CD8+ T cells were much more susceptible to apoptosis and cell death upon treatment with aCD3 F(ab)2, venetoclax, and / or aTIM-1 mAb, especially naive and central memory subsets (Fig. 1C). The combination of aCD3 F(ab)2, venetoclax, and aTIM-1 mAb also increased Treg / conventional T cell (Tcon) ratio (Fig. 10) and CD8-SIRPo+ regulatory DC subset (DC2) / CD8+ T ratio, without significantly affecting GR1+CD11b+ myeloid-derived suppressor cells (MDSCs) or B cells (Fig. 8A). This supports that the triple combination is synergistic and potentially helpful for “immune reset” and restoration of tolerance pathways.

[0180]

[0156] Tissue resident and autoimmune recipient T cells may be a significant barrier to the engraftment of donor hematopoietic grafts. \Ne next evaluated the lymphodepletion capacity of aCD3 F(ab)2 with venetoclax, and aTIM-1 treatment in vivo. We treated B6 mice with venetoclax (100 mg / kg) on days -3 and -2, aCD3 F(ab)2 (50 pg / mouse) from days -6 to -2, and the aTIM-1 (400 pg / mouse) on days -4 and -1 . On day 0, lymphodepletion was assessed in the spleen, and bone marrow (BM) using flow cytometry (Fig. 1 D). Mice receiving the combination of aCD3 F(ab)2 and venetoclax, exhibited profound T cell depletion, particularly within the CD8+ compartment in both the spleen and BM (Fig. 1 E). The combination therapy primarily depleted naive and central memory T cells while sparing effector memory T cells (Fig. 8B). Additionally, we observed a proportional increase in the Treg / Tcon ratio in both the spleen and BM enhanced with the addition of aTIM-1 mAb (Fig. 1 E). We found that combination therapy also increased the DC2 cell subset in vivo, as we observed in vitro without affecting the B cells (Fig. 8C, D). Thus, combination therapy effectively depletes recipient T cells while enriching for tolerogenic cell populations.

[0181]

[0157] Venetoclax and aCD3 F(ab')2 conditioning combined with total lymphoid irradiation enhances CD8+T cell depletion, Treg expansion, and sustained mixed chimerism following BMT. The combination of total lymphoid irradiation (TLI) and ATG serum (ATS) has long been regarded as a nonmyeloablative gold standard for inducing tolerance in combined HSCT and organ transplantation by sustained mixed chimerism and reducing GVHD. To evaluate how aCD3 F(ab)2 and venetoclax conditioning compares to the ATS depletion, we conditioned B6 mice with ATS alone, or aCD3 F(ab)2 alone, or in combination of venetoclax and aTIM-1 mAb, followed by TLI (Fig. 2A). Allogeneic BALB / c BM cells were transplanted (day 0) and we monitored peripheral blood chimerism.

[0182]

[0158] T cell depletion was comparable in the group treated with the combination of aCD3 F(ab)2 and venetoclax as in the TLI / ATS-treated group, and CD8+ T cell depletion persisted over time (Fig. 2B, C). Importantly, on day 0, Tregs were 48% in the aCD3 F(ab)2 andvenetoclax group compared to 0% in the ATS and 12% in the aCD3 F(ab)2 alone groups (P <.0001). The addition of aTIM-1 mAb boosted Tregs to 58% of CD4+ T cells (Fig. 2B). This resulted in very different CD8:Treg ratio in the different groups: 0 for ATS, 0.59 for aCD3 F(ab)2 alone, 3.8 for aCD3 F(ab)2 plus venetoclax, and 9.6 for aCD3 F(ab)2, venetoclax, and aTIM-1 (Fig. 2B). The combination therapy primarily depleted CD8+ naive and central memory T cells while sparing effector memory T cells (Fig. 9B). On day 0, B cells, MDSCs, SIRPa+ DC2, and CD11b+levels were similar among all treatment groups (Fig. 2B, Fig. 9A). On day 14 and day 30, there was a B220+ cell enrichment in the aCD3 F(ab)2 group and venetoclax group (Fig. 90).

[0183]

[0159] The day +14 peripheral blood donor chimerism levels were higher in the aCD3 F(ab)2 plus venetoclax group (40.4% total, 34.8% T cell, 38% B cell, and 62.6% myeloid cell chimerism) than the aCD3 F(ab)2 only group (14.2% total, P <.01 ; 9.1% T cell; P < .001 ; 9.4% B cell, P < .01 ; and 21 .4% myeloid chimerism, P < .01) and comparable to those observed in the TLI / ATS conditioning group (35.8% total, 32.5% T cell, 34% B cell, and 50.8% myeloid chimerism) (Fig. 2D). The addition of aTIM-1 did not enhance overall donor chimerism.

[0184]

[0160] At +90 days post-BMT, cell subsets as well as donor chimerism levels were assessed in the spleen and BM. Similar to the blood, in the BM, chimerism levels were higher in the aCD3 F(ab)2 and venetoclax than the aCD3 F(ab)2 only group (Fig. 2D) and comparable to those observed in the TLI / ATS conditioning group (Fig. 2E, Fig. 9D). In the spleen, donor chimerism levels were higher in the aCD3 F(ab)2 and venetoclax compared to the aCD3 F(ab)2 only group and comparable to TLI / ATS in all the cell subsets examined (Fig. 2F and Fig. 9D). Importantly, this effect was augmented with the addition of aTIM-1 mAb in both spleen and BM (Fig. 2E, F). Donor Tregs chimerism was increased in the aCD3 F(ab’)2, venetoclax, and / or aTIM-1 mAb groups compared to the TLI / ATS group (Fig. 2E, F). These findings suggest that the combination of venetoclax and aCD3 F(ab)2 is not only comparable to TLI / ATS, but even superior in enhancing immune tolerance, given the preferential increased donor Tregs.

[0185]

[0161] Combination of venetoclax and aCD3 F(ab’)2 conditioning with low-dose TBI enhances lymphodepletion and promotes immunoregulatory cell subsets, resulting in persistent mixed chimerism following BMT. We hypothesized that more effective lymphodepletion and the induction of immunoregulatory cells with aCD3 F(ab)2 and venetoclax therapy would facilitate donor engraftment, reducing the need for radiation or chemotherapy-based conditioning. To test this, we evaluated venetoclax and aCD3 F(ab)2 in combination with low levels of TBI (250 cGy). B6 mice were conditioned with either aCD3 F(ab)2 (50 pg / mouse) alone or a combination with venetoclax (100 mg / kg) and aCD3 F(ab)2 (50 pg / mouse) and low-dose TBI (250 cGy), followed by transplant with allogeneic BALB / c BM cells (Fig. 3A). An additionalgroup received the antagonistic aTIM-1 mAb (400 pg / mouse). Control mice were treated with ATS or Fc competent full-length aCD3 mAb (2C11 ; 5 pg / mouse).

[0186]

[0162] When mice were conditioned with aCD3 F(ab)2, venetoclax, and aTIM-1 , they showed significantly fewer T cells (9.5%) and CD8+ T cells (32.9%) in the peripheral blood at day 14, as compared to 22.2% of T cells (P < .001) and % 67.1 CD8+ T cells (P < .0001) in aCD3 F(ab)2 alone treated mice and 20.3% T cells (P < .001) and 55% CD8+ T cells (P < .001) in ATS treated mice and 25.9 % T cells (P < .0001) and 59.1% CD8+ T cells (P < .001) in Fc competent aCD3 alone treated group (Fig. 3B). Like-wise, combined treatment venetoclax, aCD3 F(ab)2, and aTIM-1 resulted in significantly enriched Treg (5.2%), compared to 2.9% Treg (P < .001) in aCD3 F(ab)2 alone and 2.9% Treg (P < .05) in ATS and 3% Treg (P < .05), in CD3 group alone (Fig. 3B).

[0187]

[0163] A similar significant trend was observed in the MDSCs. These differences in cell subtypes under the different conditioning regimens was sustained at day 30 (Fig. 3C). Additionally, we observed increased levels of total, T cell, CD8+ T, Treg, B cell and myeloid donor chimerism in the combination-treated group compared to those receiving aCD3 F(ab)2 alone, ATS, or aCD3 mAb, which persists over time (Fig. 3D). We also detected donor chimerism in the spleen and BM of the mice treated with the combination of venetoclax, aCD3 F(ab)2, and aTIM-1 mAb (Fig. 10). These findings suggest that combining venetoclax and aCD3 F(ab)2 with low-dose TBI enhances lymphodepletion and facilitates persistent donor engraftment, while simultaneously promoting a tolerogenic environment.

[0188]

[0164] Venetoclax and aCD3 F(ab’)2 conditioning induced sustained mixed chimerism and immune tolerance to allogeneic islets. We next investigated the effects of venetoclax and aCD3 F(ab)2 conditioning on alloreactivity and the long-term survival of pancreatic islet transplants. B6 mice were conditioned with either aCD3 F(ab)2 alone, venetoclax and aCD3 F(ab)2, or venetoclax, aCD3 F(ab’)2, and aTIM-1 mAb, along with low-dose TBI (250 cGy). We administered allogeneic BALB / c BM cells on day 0, and 100 firefly luciferase-expressing BALB / c is-lets were transplanted under the kidney capsule on day 1 (Fig. 4A). Islet survival was monitored using BLI, and cell sub-sets and chimerism levels were also monitored. Conditioning with venetoclax, aCD3 F(ab)2, and aTIM-1 resulted in deep T cell lymphodepletion, especially ofthe CD8+ T cells and increased frequency of tolerogenic Tregs and CD8-SIRPa+ DCs as early as 14 days post-BMT (Fig. 4B). CD8+ T cell depletion and upregulation of CD8-SIRPa+ DCs were sustained at day +30. We observed high levels of donor chimerism in the mice treated with venetoclax and aCD3 F(ab)2, and this further increased when adding aTIM-1 (Fig. 4C). Islet allograft survival was achieved in both these groups (Fig. 4D, E). These findings highlight the therapeutic potential of this conditioning strategy for transplantation immune tolerance.

[0165] Venetoclax and aCD3 F(ab’)2 treatment following allogeneic BMT prevents GVHD. PTCy depletes highly proliferative alloreactive T cells, whereas Tregs are thought to be resistant. However, PTCy can expose patients to additional risk, such as organ toxicity that may increase treatment-related mortality. To evaluate the potential use of posttransplant aCD3 F(ab)2 plus venetoclax and (PT-aCD3Fab + Ven) to prevent GVHD, we utilized a fully major histocompatibility complex (MHC) mismatched BMT model. We reasoned that the combination treatment over just 2 days, akin to PTCy, would act to preferentially deplete alloreactive T cells and promote Tregs to prevent GVHD. We administered allogeneic BM and T cells from MHC major mismatched B6 (H-2b) donors to BALB / c (H-2d) recipient mice. Donor T cells were isolated from DTR-Foxp3+Luc+GFP+ B6 (H-2b) mice, allowed for BLI. BALB / c mice received venetoclax on days 3 and 4, and aCD3 F(ab)2 (50 pg / mouse) on day 3 post- BMT. Control groups were treated with either venetoclax or aCD3 F(ab)2 as single agents (Fig. 5A).

[0189]

[0166] Mice receiving PT-aCD3Fab + Ven demonstrated significantly improved survival rates and lower GVHD scores compared to those treated with either agent alone (P = .01) (Fig. 5B, C, Fig. 11). Notably, Tregs expanded in mice treated with PT-aCD3Fab + Ven, as evidenced by BLI (Fig. 5D, E), and were detected in circulation by day 21 (Fig. 5F). Although an initial expansion of Tregs was observed on day 6 post-BMT in the Tcon group likely driven by the inflammatory microenvironment associated with GVHD and T cell activation the ratio of Tregs to Tcons was notably higher in the PT-CD3 Fab + Ven group which likely underlies the GVHD prevention observed (Fig. 5G).

[0190]

[0167] Venetoclax and aCD3 F(ab’)2 treatment following BMT protects against GVHD and exhibits reduced toxicity and inflammatory response compared to PTCy. To investigate how the combination of venetoclax and aCD3 F(ab)2 compares directly to PTCy, BALB / c (H-2d) mice received allogeneic BM and T cells from MHC major mismatched B6 (H-2b) donors. On days 3 and 4 posttransplant, the mice were treated with either the combination of venetoclax plus aCD3 F(ab)2, aCD3 F(ab)2 alone, or PTCy (Fig. 6A). We found that mice treated with either the combination of PT-aCD3Fab + Ven or PTCy exhibited fewer symptoms of GVHD and had improved survival (Fig. 6B, C, Fig. 12). On day +6, PTCy treatment led to more profound T cell lymphodepletion, partic-ularly of CD8+ T cells, than aCD3 F(ab)2 groups but also showed a much greater reduction in both the overall number and proportion of T regs after HSCT (Fig. 6D).

[0191]

[0168] PTCy is associated with the frequent occurrence of peri-engraftment inflammation that can result in organ toxicity. To test if there are any early inflammatory states between approaches, serum was collected from these experimental mice and analyzed for cytokine and chemokine concentrations using a multiplex assay (Luminex). We observed elevatedserum levels of interleukin (IL)-2 in mice treated with PT-aCD3Fab + Ven compared to those treated with PTCy, likely due to the activation of T cells by aCD3Fab (Fig. 6E). The increased IL-2 levels may contribute to the higher Treg and CD8+ T cell levels observed with PT-aCD3 Fab + Ven group. In contrast, IL-22, MIP-1a, and IL-6 were significantly higher in the serum of PTCy-treated mice compared to PT-aCD3Fab + Ven mice and at levels comparable to mice receiving only Tcons (Fig. 6E). Moreover, Ml P-1 p levels were significantly lower in the serum of PT-aCD3Fab + Ven mice compared to both PTCy and Tcons receiving mice (Fig. 6E).

[0192]

[0169] To assess for potential organ toxicity, we measured serum markers of kidney, liver, and cardiac function. Serum creatinine was significantly elevated in the PTCy group (0.278 mg / dL) compared to the PT-aCD3Fab + Ven group (0.228 mg / dL, P = .04) (Fig. 6F). The same was true for alanine transaminase in the PTCy group (117 U / L) compared to the PT-CD3 Fab / Ven group(77.6 U / L, P = .02). These results suggest that targeted T cell depletion using mAb approaches might be less toxic than chemotherapy approaches.

[0193]

[0170] Comparison of posttransplant depletion strategies for islet allograft tolerance with full donor chimerism. Like TLI / ATS conditioning, PTCy has been used in clinical trials of combined organ and HSCT strategies for the induction of immune tolerance. Both approaches result in mixed donor chimerism and organ tolerance, but if full donor chimerism is achieved, it can lead to GVHD. As a practical translational consideration, we evaluated the 2 strategies in an islet trans-plantation model in which full donor chimerism is achieved to evaluate the risk of GVHD and if there were differences in islet allograft survival (Fig. 7A).

[0194]

[0171] B6 mice were conditioned with either aCD3 F(ab)2, venetoclax plus aCD3 F(ab)2, or a triple combination of venetoclax, aCD3 F(ab)2, and aTIM-1 . All groups received low-dose TBI. On day 0, the mice received allogeneic BALB / c BM cells, and on day 1 , 100 firefly luciferase-expressing BALB / c islets were trans planted under the kidney capsule. As part of the prophylactic strategy, mice received a single dose of aCD3 F(ab)2 on day 4, and venetoclax on days 3 and 4, or 2 doses of cyclophosphamide.

[0195]

[0172] PTCy treatment resulted in greater islet loss (45 x 104 ps-1 mouse-1), whereas PT- aCD3Fab + Ven treatment maintained islet mass (97.7 x 104 ps-1 mouse-1), integrity and survival, as shown in Figure 7B, C. Both PTCy and the PT-aCD3 + Ven regimen successfully induced full donor chimerism (Fig. 7D) and no GVHD after 60 days post-HSCT, indicating that either approach is effective for GVHD prevention in various trans-plantation settings. The loss of the islets may be due to direct toxicity of the cyclophosphamide or possibly due to increased local inflammation, which is known to adversely affect islet function and survival. Notably, the PTCy group had elevated serum levels of MCP-1 (15.1 pg / mL) compared to PT-aCD3Fab + Ven (8.5 pg / mL) (P = .007, 1-way ANOVA test) (Fig. 7E). High MCP-1 levels are associated with worse islet survival following allogeneic HSCT. These findings highlight the potential ofPT-aCD3Fab + Ven as a promising therapeutic strategy to reduce to risk of GVHD while establishing immune tolerance.

[0196]

[0173] In this study, we explored a novel immune conditioning approach that takes advantage of apoptosis by aCD3 F(ab)2synergized with anti-BCL-2 and aTIM-1 therapy. We showed that this unique combination could be effectively used for more complete T cell depletion and induction of regulatory pathways, with improvements in donor engraftment, GVHD prevention, and tolerance to concomitantly transplanted organ or tissues with islets as a test case in this study.

[0197]

[0174] As compared to aCD3 therapy alone, the combination of venetoclax and aCD3 F(ab)2 strongly induces apoptosis in T cells, particularly in naive and central memory CD8+ T cells. This approach also enriched Tregs and regulatory CD8-SIRPa+ DCs. Because recipient CD8+ T cells represent a major barrier to donor engraftment, their efficient depletion may explain the more successful donor engraftment observed. Venetoclax itself is being integrated into HSCT conditioning with evidence for excellent tolerability and facilitation of engraftment, possibly by direct action in HSCs. The addition of TIM-1 blockade augments engraftment, particularly of Tregs. Anti-TIM1 therapy alone given pre-HSCT in preclinical models can prevent GVHD if administered during conditioning, but the concept of using it with selective aCD3 F(ab)2 and venetoclax depletion post-transplant has not been investigated.

[0198]

[0175] PTCy is associated with significant adverse effects, including cardiopathology, neurotoxicity, recurrent infections, and infertility. Furthermore, PTCy negatively impacts thymic mi-grants and can hinder donor Treg engraftment in preclinical HSCT models. The use of PTCy also has limited value in nonmalignant conditions where toxicities should be minimized. Our results suggest that PT-aCD3Fab + Ven could substitute PTCy with less toxicity. GVHD prevention was comparable, but the PT-aCD3Fab + Ven mice showed significantly less peritreatment organ toxicity and a different serum cytokine profile (increased IL-2, and decreased IL-6, MIP-1a, IL-22), which can contribute to engraftment syndrome, cytokine release, and dysregulated immunity. These results favor further translational investigation in the treatment of immuno-deficiencies, autoimmunity, and tolerance induction.

[0199]

[0176] We evaluated MHC major mismatched islet transplantation models to investigate how aCD3 F(ab)2 and venetoclax treatment might contribute to immune tolerance, including an evaluation of PT-aCD3Fab + Ven. The use of venetoclax has been explored in tolerance induction, and for islet transplantation in particular, this may be advantageous, as it has been shown to prevent diabetes in non-obese diabetic (NOD) mice by eliminating senescent beta cells, a significant contributor to T1 D pathogenesis. In contrast, in our murine models, the use of PTCy was highly toxic to islets, possibly directly or more likely because of the production ofMCP-1 and other inflammatory cytokines known to be toxic to islets but also implicated in damaging the kidney in combination with HSCT studies. In addition, in the context of islet trans-plantation, PTCy treatment has been shown to induce T1D in NOD mice by reducing Tregs, leading to questions about how well PTCy might perform comparably in patients with autoimmune conditions.

[0200]

[0177] In summary, the novel aCD3-based conditioning approaches reported here modulate recipient immunity to overcome engraftment barriers and induce tolerance. This regimen has the potential to replace current strategies involving ATG and chemotherapy agents like cyclophosphamide to facilitate less toxic and more effective HSCT, especially for nonmalignant indications. In human combined kidney and HSCT, the addition of aCD3 and venetoclax may improve donor chimerism and reduce the irradiation-based preconditioning burden for patients. Furthermore, the addition of a stem cell-depleting agent, such as anti-c- kit mAb, could allow for the removal of chemoradiotherapy altogether.

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[0263]

[0225] Each publication cited in this specification is hereby incorporated by reference in its entirety for all purposes.

[0264]

[0226] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims

[0265]

[0227] As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the culture" includes reference to one or more cultures and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

Claims

What is Claimed is:1 . A method for lymphodepletion of a mammalian host, the method comprising: administering a conditioning regimen comprising a combination of an effective dose of (i) an antibody specific for CD3 and (ii) an effective dose of a BCL2 inhibitor to a recipient.

2. The method of claim 1 , wherein the administering results in apoptosis of T cells present in the recipient.

3. The method of claim 1 or claim 2, wherein the administering results in an increase in the number of one or more of regulatory (Treg) cells, myeloid derived suppressor cells (MDSC), and tolerogenic dendritic cells (DC2).

4. The method of any of the previous claims, further comprising:administering an effective dose of an immunosuppressive regimen to the recipient.

5. The method of claim 4, wherein the immunosuppressive regimen comprises administration of an effective dose of an antibody specific for TIM1.

6. The method of claim 4, wherein the immunosuppressive regimen comprises administration of total lymphoid irradiation.

7. The method of claim 4, wherein the immunosuppressive regimen comprises administration of a low dose total body irradiation.

8. The method of any of the previous claims, where the method is performed in the absence of administration of anti-thymocyte globulin (ATG) or cyclophosphamide.

9. The method of any of the previous claims wherein the mammalian host is human.

10. The method of claim 9, wherein the antibody specific for CD3 specifically binds to human CD3.11 . The method of any of the previous claims, wherein the antibody specific for CD3 lacks an Fc region sequence.

12. The method of any of the previous claims, wherein the antibody specific for CD3 comprises an Fc sequence modified to reduce binding to high affinity FcyR.

13. The method of any of the previous claims, wherein the antibody specific for CD3 is an F(ab)2 fragment, or an F(ab’)2 fragment.

14. The method of any of the previous claims, wherein the antibody specific for CD3 comprises a variable region sequence of muromonab-CD3, otelixizumab, teplizumab, foralumab, or visilizumab.

15. The method of any of the previous claims, wherein the BCL2 inhibitor is venetoclax (ABT- 199).

16. The method of any of the previous claims, wherein the BCL2 inhibitor is navitoclax (ABT-263); S55746 (BCL201); Obatoclax (GX15-070); or gossypol.

17. The method of any of the previous claims wherein the conditioning regimen is administered prior to bone marrow transplantation.

18. The method of any of the previous claims wherein the conditioning regimen is administered prior to hematopoietic stem cell transplantation.

19. The method of any of the previous claims wherein the conditioning regimen is administered prior to CAR-T cell administration.

20. The method of any of the previous claims wherein the conditioning regimen is administered prior to solid organ transplantation, where the solid organ transplantation is optionally combined with BMT or HSCT.21 . The method of any of the previous claims wherein the conditioning regimen is administered for the treatment of GVHD, where administration may be prior to, or following transplantation of hematopoietic cells.

22. The method of claim 21 , wherein administration follows a determination of GVHD.

23. The method of any of the previous claims wherein the conditioning regimen is administered for the treatment of an autoimmune disease.

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