Methods of using LFA3-FC fusions for cell therapy

Abstract

The present disclosure provides a method of treating an autoimmune disease or inflammatory disease in an individual comprising administering to the individual a combination therapy. The combination therapy comprises a CD-2 binding molecule comprising a CD2-binding domain linked to an Fc domain and a composition comprising modified regulatory T (Treg) cells. The CD-2 binding molecule is administered first to precondition the individual for administration of the composition comprising the modified Treg cells.

Description

Docket No.: 237752001540METHODS OF USING LFA3-FC FUSIONS FOR CELL THERAPYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 692,527, filed September 9, 2024, which is incorporated herein by reference in its entirety.REFERENCE TO A SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (237752001540SEQLIST.xml; Size: 6,074 bytes; and Date of Creation: August 29, 2025) is herein incorporated by reference in its entirety.FIELD

[0003] The present disclosure relates, in some aspects, to combination therapies comprising CD-2 binding molecules and compositions comprising modified Treg cells for use in treating autoimmune disease or inflammatory disease.BACKGROUND

[0004] Regulatory T (Treg) cells have the potential to treat various autoimmune and inflammatory diseases. Recent studies have shown the therapeutic potential of Treg-related immune therapies, including low dose interleukin-2 (IL-2) and IL-2 muteins, in a variety of autoimmune settings. Clinical studies have also evaluated the potential of polyclonal Treg cells as a treatment option for organ transplant recipients and patients suffering from autoimmune conditions. Although the Treg cell infusions were well tolerated, only limited efficacy was observed. A significant challenge in advancing Treg cell therapies for treating autoimmune and inflammatory disease lies in the resistance of effector T (Teff) cells to Treg- mediated suppression. The predominance of these highly activated Teff cells is correlated to loss of immune system balance. Accordingly, there is an unmet need for novel, safe, and effective therapies that can limit autoimmune or inflammatory related tissue destruction. Provided herein are combination therapies comprising modified Treg cells and LFA3 molecules that meet such needs.1MOFO-360045222Docket No.: 237752001540BRIEF SUMMARY

[0005] The present disclosure is directed to combination therapies that comprise administering: a) CD-2 binding molecules comprising a CD2-binding domain of human leukocyte function-associated antigen 3 (LFA3) linked to an Fc domain and b) compositions comprising modified regulatory T (Treg) cells.

[0006] In some aspects, provided herein is a method of treating an autoimmune disease or inflammatory disease in an individual comprising administering to the individual: a) about 1 mg to about 30 mg of a CD-2 binding molecule comprising a CD2 -binding domain of human leukocyte function-associated antigen 3 (LFA3) linked to an Fc domain; and b) a composition comprising modified regulatory T (Treg) cells, wherein the modified Treg cells express a therapeutic protein; and wherein the composition comprising the modified Treg cells is administered after administration of the CD-2 binding molecule.

[0007] In some embodiments, about 3 mg to about 10 mg of the CD-2 binding molecule is administered to the individual. In some embodiments, about 3 mg of the CD-2 binding molecule is administered to the individual. In some embodiments, about 5 mg of the CD-2 binding molecule is administered to the individual. In some embodiments, about 7 mg of the CD-2 binding molecule is administered to the individual. In some embodiments, about 10 mg of the CD-2 binding molecule is administered to the individual.

[0008] In some embodiments, the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, about 3 mg, 5 mg, 7 mg, or 10 mg of the CD-2 binding molecule is administered to the individual, and the CD-2 binding molecule is administered by intramuscular injection.

[0009] In some embodiments, the CD-2 binding molecule is administered intravenously. In some embodiments, about 3 mg of the CD-2 binding molecule is administered to the individual and the CD-2 binding molecule is administered intravenously.

[0010] In some embodiments, the LFA3 domain comprises an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the LFA3 domain comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the Fc domain comprises an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the Fc domain comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD-2 binding molecule comprises an amino acid sequence with at least about 90% identity to the2MOFO-360045222Docket No.: 237752001540 amino acid sequence of SEQ ID NO: 2. In some embodiments, the CD-2 binding molecule comprises the amino acid sequence of SEQ ID NO: 2.

[0011] In some embodiments, the composition comprising the modified Treg cells is administered about 50 days to about 120 days after administration of the CD-2 binding molecule. In some embodiments, the composition comprising the modified Treg cells is administered about 80 days to about 90 days after administration of the CD-2 binding molecule.

[0012] In some embodiments, administration of the CD-2 binding molecule to the individual results in a reduction of CD4+ effector memory T-cells (Tern) in the peripheral blood of the individual. In some embodiments, the composition comprising the modified Treg cells is administered when the level of CD4+ Tern in the peripheral blood of the individual is reduced by at least about 40% compared to the level of CD4+ Tern in the peripheral blood of the individual prior to administration of the CD-2 binding molecule. In some embodiments, the composition comprising the modified Treg cells is administered when the level of CD4+ Tem in the peripheral blood of the individual is reduced by about 25% to about 50% compared to the level of CD4+ Tern in the peripheral blood of the individual prior to administration of the CD-2 binding molecule.

[0013] In some embodiments, administration of the CD-2 binding molecule to the individual results in a reduction of CD4+ central memory T-cells (Tcm) in the peripheral blood of the individual. In some embodiments, the composition comprising the modified Treg cells is administered when the level of CD4+ Tcm in the peripheral blood of the individual is reduced by at least about 40% compared to the level of CD4+ Tcm in the peripheral blood of the individual prior to administration of the CD-2 binding molecule. In some embodiments, the composition comprising the modified Treg cells is administered when the level of CD4+ Tcm in the peripheral blood of the individual is reduced by about 30% to about 50% compared to the level of CD4+ Tcm in the peripheral blood of the individual prior to administration of the CD-2 binding molecule.

[0014] In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the serum of the individual is less than about 50 ng / mL. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the serum of the individual is less than about 20 ng / mL. In some embodiments, the3MOFO-360045222Docket No.: 237752001540 composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the serum of the individual is about 10 ng / mL to about 50 ng / mL. In some embodiments, the composition comprising the modified Treg cells is administered intravenously.

[0015] In some embodiments, about 3 mg of the CD-2 binding molecule is administered to the individual, wherein the CD-2 binding molecule is administered by intramuscular injection, and wherein the composition comprising the modified Treg cells is administered about 65 days to about 80 days after administration of the CD-2 binding molecule. In some embodiments, about 5 mg of the CD-2 binding molecule is administered to the individual, wherein the CD-2 binding molecule is administered by intramuscular injection, and wherein the composition comprising the modified Treg cells is administered about 75 days to about 95 days after administration of the CD-2 binding molecule. In some embodiments, about 7 mg of the CD-2 binding molecule is administered to the individual, wherein the CD-2 binding molecule is administered by intramuscular injection, and wherein the composition comprising the modified Treg cells is administered about 80 days to about 105 days after administration of the CD-2 binding molecule. In some embodiments, about 10 mg of the CD-2 binding molecule is administered to the individual, wherein the CD-2 binding molecule is administered by intramuscular injection, and wherein the composition comprising the modified Treg cells is administered about 85 days to about 120 days after administration of the CD-2 binding molecule. In some embodiments, about 3 mg of the CD-2 binding molecule is administered to the individual, wherein the CD-2 binding molecule is administered intravenously, and wherein the composition comprising the modified Treg cells is administered about 55 days to about 80 days after administration of the CD-2 binding molecule.

[0016] In some embodiments, therapeutic protein is a T cell receptor (TCR) comprising an antigen-binding site that specifically binds to antigen. In some embodiments, the therapeutic protein is a chimeric antigen receptor (CAR). In some embodiments, the modified Treg cells are CD4+, CD25+ and CD1271o. In some embodiments, the CAR comprises an antigen binding domain that specifically binds to an antigen that is associated with an autoimmune disease or inflammatory disease. In some embodiments, the antigen is selected from the group consisting of MHC class I, MHC class II, collagen type V, K alpha 1 tubulin, MHC class I related chain A (MICA), fibronectin, collagen type IV, collagen type VI, vimentin, angiotensin II type 1 receptor (AGTR1), perlecan, agrin, collagen type I, collagen type II,4MOFO-360045222Docket No.: 237752001540 collagen type III, cardiac myosin, an islet cell autoantibody (ICA) antigen, insulin, glutamic acid decarboxylase (GAD), myelin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, astrocyte proteins, glial fibrillary protein (GFAP), SlOObeta, beta cell antigen, insulin B chain, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD65), islet-associated antigen 2, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), zinc transporter 8 (ZnT8), islet antigen 2 (IA-2), heat shock protein 60 (HSP60), chromogranin A, dnaJ (heat shock protein), citrullinated- vimentin, human cartilage glycoprotein-39, mitochondrial component, pyruvate dehydrogenase (mitochondrial), E2 component of pyruvate dehydrogenase, E2 component of branched chain 2-oxo acid dehydrogenase, E2 component of 2-oxo-glutarate dehydrogenase complex, E3 binding protein of dihydrolipoamide dehydrogenase, a nuclear component, nuclear protein splOO, nuclear pore complex protein gpl20, centromere, acetylcholine receptor (AChR), aquaporin-4 (AQP-4), CTLA-4, ICAM, LFA-3, CD40 / CD154, ICOS / ICOSL, CD52, nuclear factor of activated T cells (NF AT), phospholipase C (PLC), CD25, Janus kinase, B cell activating factor (BAFF), a proliferating inducing ligand (APRIL), IL6R, IL 17, IL12 / IL23, an integrin, a sphingosin receptor, melanocyte antigen, tolllike receptor, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, MyD88, an IL-lR-associated kinase (IRAK), an allergen associated with the allergic disorder, a citrullinated protein or citrullinated fragments thereof, citrullinated vimentin, and citrullinated filaggrin.

[0017] In some embodiments, the antigen binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof. In some embodiments, the antigen binding domain of the CAR specifically binds to citrullinated vimentin. In some embodiments, the antigen binding domain of the CAR binds to all three of (i) citrullinated vimentin, (ii) citrullinated filaggrin, and (iii) citrullinated fibrinogen, or citrullinated peptides fragments thereof, optionally wherein the antigen binding domains further binds to citrullinated tenascin C.

[0018] In some embodiments, the modified Treg cells are human T cells. In some embodiments, the modified Treg cells are primary T cells.

[0019] In some embodiments, the individual is a human. In some embodiments, the autoimmune disease or inflammatory disease is a disease mediated by CD2. In some embodiments, the autoimmune disease or inflammatory disease is selected from the group consisting of type I diabetes, rheumatoid arthritis, hidradenitis suppurativa, amyotrophic lateral sclerosis, celiac disease, and inflammatory bowel disease.5MOFO-360045222Docket No.: 237752001540

[0020] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. These and other embodiments of the invention are further described by the detailed description that follows.DESCRIPTION OF THE FIGURES

[0021] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0022] The drawings illustrate certain embodiments of the features of this disclosure. These embodiments are not intended to limit the scope of the claims in any manner.

[0023] FIG. 1 illustrates an overview of the study design. The study evaluated single ascending doses (SAD) of the study drug (LF-vl) or matching placebo administered to five cohorts of healthy participants. Sentinel participants were dosed in each cohort to assess the acute safety of study drug (LF-vl or matching placebo) prior to enrolling the rest of the cohort. The dosage for cohorts 1 through 4 were 1 mg, 3 mg, 10 mg, and 30 mg, respectively. The dosage for cohort 5 was based on an analysis of safety and pharmacokinetics (PK) and pharmacodynamic (PD) data from the first 4 cohorts.

[0024] FIG. 2 shows the study design representative for cohorts 1 through 4.

[0025] FIGS. 3A-3B show mean (± standard deviation (SD)) serum concentration of LF-vl versus time following a single intramuscular (IM) or intravenous (IV) administration of LF- vl on linear scale (FIG. 3A) and semi-log scale (FIG. 3B). The lower limit of quantitation (LLOQ) for LF-vl is 0.005 pg / mL, and all values below LLOQ were set to LLOQ / 2. All serum LF-vl concentrations were below the limit of quantitation (BLQ) in the placebo IM and placebo IV group. Mean concentration profiles of the placebo groups are not displayed.

[0026] FIGS. 4A-4D show longitudinal average subset graphs showing the trends and variations in the levels of T cell subsets in samples of peripheral blood from the study participants in Cohorts 1-5 from baseline throughout the treatment period. Shown in FIG. 4A are the levels of CD4+effector memory T (Tem) cells. Shown in FIG. 4B are the levels of CD4+central memory cells (Tcm). Shown in FIG. 4C are the levels of regulatory T (Treg) cells. Shown in FIG. 4D are the levels of CD4+naive T (Tn) cells.6MOFO-360045222Docket No.: 237752001540

[0027] FIGS. 5A-5L show the decreases in absolute cell counts observed following 1 mg to 30 mg IM administration of the study drug. Shown are the mean (+SD) percent change from baseline (PCFB) of absolute count of CD4+Tcmcells on a numeric (FIG. 5A) and ordinal (FIG. 5B) scale, of absolute count of CD4+Temcells on a numeric (FIG. 5C) and ordinal (FIG. 5D) scale, of absolute count of Tregcells on a numeric (FIG. 5E) and ordinal (FIG. 5F) scale, of absolute count of CD4+Tnaive cells on a numeric (FIG. 5G) and ordinal (FIG. 5H) scale, of absolute count of CD8+Temra cells on a numeric (FIG. 51) and ordinal (FIG. 5J) scale, and of absolute count of NK cells on a numeric (FIG. 5K) and ordinal (FIG. 5L) scale.

[0028] FIGS. 6A-6B depict linear scales of the mean (+SD) percent change from baseline (PCFB) of CD4+cell ratios (Treg: CD4+Tcmratio (FIG. 6A) and Treg: CD4+Temratio (FIG. 6B)) over time by type and treatment (placebo or study drug).

[0029] FIGS. 7A“71 I depict Eavg, 3-36 of different cell populations following placebo or study drug treatment Eavg, 3-36 of CD3+ T cells (FIG. 7 A); Eavg, 3-36 of CD4+ T cells (FIG. 7B); Eavg, 3-36 of Treg cells (FIG. 7C); Eavg, 3-36 of CD4+ Tnaive cells (FIG. 7D); Eavg, 3-36 of CD4+ Tcm cells (FIG. 7E); Eavg, 3-36 of CD4+ Tem cells (FIG. 7F); Eavg, 3-36 of CD8+ T cells (FIG. 7G); Eavg, 3-36 of CD8+ Tnaive cells (FIG. 7H); Eavg, 3-36 of CD8+ Tcm cells (FIG. 71); Eavg, 3-36 of CD8+ Tem cells (FIG. 7 J); Eavg, 3-36 of CD8+ Temra cells (FIG. 7K); Eavg, 3-36 of NK cells (FIG. 7L); Eavg, 3-36 of B cells (FIG. 7M).

[0030] FIGS. 8A-8D depict Eavg, 3-36 of different cell populations following placebo or study drug treatment: Eavg, 3-36 of Treg : CD4+ Tcm ratio (FIG. 8A); Eavg, 3-36 of Treg : CD4+ Tem ratio (FIG. 8B); Eavg, 3-36 of Treg : CD8+ Tcm ratio (FIG. 8C); Eavg, 3-36 of Treg : CD8+ Tem ratio (FIG. 8D).

[0031] FIGS. 9A-9D depict Eavg, 3-36 for different cell population ratios following placebo or study drug treatment: Eavg, 3-36 of Treg : CD4+ Tcm ratio (FIG. 9A); Eavg, 3-36 of Treg : CD4+ Tem ratio (FIG. 9B); Eavg, 3-36 of Treg : CD8+ Tcm ratio (FIG. 9C); Eavg, 3-36 of Treg : CD8+ Tem ratio (FIG. 9D).

[0032] FIGS. 10A-10C show the stratification of T cell depletion into three phases following administration of LF-vl. FIG. 10A provides a schematic overview of the stages of cell depletion from Hour 2 to Day 3 (i.e., Redistribution Effect), Day 3 to Day 8 (i.e., Early PD), and Day 8 to Day 36 (i.e., Late PD). FIGS. 10B-10C show the levels of T cell subset depletion in samples of peripheral blood from the study participants from baseline across7MOFO-360045222Docket No.: 237752001540 multiple doses of LF-vl in Cohorts 1-5 in each stage as outlined in FIG. 10A, for CD4+Temcells (FIG. 10B) and CD4+Tcmcells (FIG. IOC).

[0033] FIGS. 11 A-l IB show Treg / Tsubset ratios for CD4+Tem(FIG. 11 A) and CD4+Tcm(FIG. 11B) over time.

[0034] FIGS. 12A-12D shows the correlation between CD2 and pharmacodynamics (PD). FIG. 12A shows baseline CD2 expression level of all T cell subsets in Cohorts 1-4. FIG. 12B shows the average % change from baseline (D3-D36) vs the baseline CD2 for all T cell subsets in Cohorts 1-5. FIG. 12C depicts baseline CD2 levels (MFI) in different T / NK cell populations. In the box plots, the solid line within the box is the median. The ends of the “box” are the 25th and 75th percentiles. These are also referred to as the first and third quartiles. The whiskers show the lowest data value still within 1.5 interquartile range (IQR) of the lower quartile, and the highest value still within 1.5 IQR of the upper quartile, where IQR is the interquartile range (the difference between the third and first quartiles, the middle 50%). FIG. 12D depicts baseline CD2 levels (MFI) vs Eavg, 3-36 (%) in T / NK cell populations. Placebo group is not included. Only the following cells subtypes are included: CD8+Tem cells, CD8+Tcmcells, CD8+ Temra cells, CD8+Tnaive cells, CD4+Tnaive cells, CD4+Tem cells, CD4+Tcm cells, Treg cells, NK cells. Due to the limit of y-axis, the following points are not displayed on the graph: Subject 014 (3mg IM) Tregcells (CD2 baseline level: 9139.85, Eavg, 3-36: 202.70), Subject 015 (3mg IM) CD8+Temra cells (CD2 baseline level: 4864.51, Eavg, 3-36: 145.65).

[0035] FIG. 13 shows the time under the depletion threshold (days) at 40% and 80%.

[0036] FIGS. 14A-14D show normalized cell number of SOI CAR Treg cells and CD4+Tem cells as a function of LF-vl concentration (pg / mL) for in vitro co-cultured samples of PBMCs and SOI CAR Treg cells. FIGS. 14A-14B show PBMCs and SOI CAR Treg cells cocultured at an effector to target ratio (E:T; PBMC:S01 CAR Treg) of 25 for donors 1-2 (FIG. 14A) and donors 3-4 (FIG. 13B). FIGS. 14C-14D show PBMCs and SOI CAR Treg cells co-cultured at an E:T ratio (PBMC:S01 CAR Treg) of 5 for donors 1-2 (FIG. 14C) and donors 3-4 (FIG. 14D)

[0037] FIG. 15 shows drug plasma concentration of the CD-2 binding molecule (e.g., LF- vl) or cell count (cells / pL) of CD4+Temcells as a function of time. The horizontal and vertical dotted lines represent the drug plasma concentration of LF-vl and the time point,8MOFO-360045222Docket No.: 237752001540 respectively, in which it is anticipated to be appropriate to administer a Treg cell therapy after LF-vl administration.

[0038] FIGS. 16A-16B show the observed (Cohorts: 1, 3, and 4; FIG. 16A) and predicted (FIG. 16B) percentage change from baseline of CD4+ Tcmcells over time across multiple doses of LF-vl. In FIG. 16B, dose amounts (1 mg, 3 mg, 5 mg, 7 mg, 10 mg) are indicated at the top of each plot.

[0039] FIGS. 17A-17B show the observed (Cohorts: 1, 3, and 4; FIG. 17A) and predicted (FIG. 17B) pharmacokinetic profiles of LF-vl across multiple doses. In FIG. 17B, dose amounts (1 mg, 3 mg, 5 mg, 7 mg, 10 mg) are labeled on the plot corresponding to each line.DETAILED DESCRIPTION

[0040] Provided herein are methods of treating an autoimmune disease autoimmune disease or inflammatory disease in an individual, comprising administering to the individual a combination therapy comprising: a) a CD-2 binding molecule comprising a CD2-binding domain of human leukocyte function-associated antigen 3 (LFA3) linked to an Fc domain and b) a composition comprising modified regulatory T (Treg) cells, wherein the modified Treg cells express a therapeutic protein, and wherein the composition comprising the modified Treg cells is administered after administration of the CD-2 binding molecule. Such diseases include type- 1 -diabetes, rheumatoid arthritis, hidradenitis suppurativa, amyotrophic lateral sclerosis, celiac disease, inflammatory bowel disease, psoriasis, or any disease, disorder, or conditions mediated by CD2.

[0041] All publications, comprising patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0042] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0043] The present disclosure relates to methods of treating an autoimmune disease or inflammatory disease in an individual by administering a combination therapy comprising: a)9MOFO-360045222Docket No.: 237752001540 about 1 mg to about 30 mg of a CD-2 binding molecule comprising a CD2-binding domain of human leukocyte function-associated antigen 3 (LFA3) linked to an Fc domain; and b) a composition comprising modified regulatory T (Treg) cells, wherein the modified Treg cells express a therapeutic protein; and wherein the composition comprising the modified Treg cells is administered after administration of the CD-2 binding molecule.

[0044] The CD-2 binding molecules provided herein bind to and modulate the activity of the transmembrane adhesion molecule CD2 (also known as LFA-2 for leukocyte function- associated antigen-2) and deplete CD2-expressing cells. Depletion of CD2-expressing cells, for example Treg-resistant effector T (Teff) cells, can help reset the microenvironment in the individual being treated, thus preconditioning the individual for administration of the composition comprising the modified Treg cells. This combination therapy may have beneficial effects in the treatment of autoimmune disease or inflammatory disease, such as diabetes, rheumatoid arthritis, hidradenitis suppurativa, amyotrophic lateral sclerosis, celiac disease, inflammatory bowel disease, and psoriatic arthritis.

[0045] In certain embodiments, the CD-2 binding molecules provided herein bind to CD2, modulate CD2 signaling, and selectively deplete CD2-expressing memory T cells (Temcells). In some embodiments, administration of the CD-2 binding molecule preconditions the individual for subsequent administration of the composition comprising the modified Treg cells.

[0046] Without wishing to be bound by theory, the CD-2 binding molecules provided herein modulate the function of and selective depletion of CD2+ Temcells to rebalance the regulatory T (Treg) / Temcell ratio. In some embodiments, rebalancing of regulatory Treg / Tem cell numbers in the individual preconditions the individual for subsequent administration of the composition comprising the modified Treg cells. In some embodiments, the CD-2 binding molecules provided herein promote elimination and / or suppression of pathogenic T effector cells. In some embodiments, elimination and / or suppression of pathogenic T effector cells in the individual preconditions the individual for subsequent administration of the composition comprising the modified Treg cells. In some embodiments, the CD-2 binding molecules provided herein modulate the interaction between CD2 and LFA3, thereby interrupting CD2- mediated T cell co-stimulation. In some embodiments, interrupting CD2-mediated T cell costimulation in the individual preconditions the individual for subsequent administration of the composition comprising the modified Treg cells. In some embodiments, the CD-2 binding molecules provided herein deplete CD2+ T cells via FcR-mediated antibody dependent10MOFO-360045222Docket No.: 237752001540 cellular cytotoxicity (ADCC). In some embodiments, depletion of CD2+ T cells via FcR- mediated ADCC in the individual preconditions the individual for subsequent administration of the composition comprising the modified Treg cells. In some embodiments, the CD-2 binding molecules provided herein deplete CD2+ T cells via apoptosis. In some embodiments, depletion of CD2+ T cells via apoptosis in the individual preconditions the individual for subsequent administration of the composition comprising the modified Treg cells. In some embodiments, the CD-2 binding molecules provided herein decrease the number of CD2hlghT memory cells (Tmem), e.g., central memory (Tcm) and effector memory (Tem) T cells, while preserving regulatory T cells (Treg). In some embodiments, decreasing the number of CD2hlghT memory cells (Tmem), e.g., central memory (Tcm) and effector memory (Tem) T cells, while preserving regulatory T cells (Treg), in the individual preconditions the individual for subsequent administration of the composition comprising the modified Treg cells. In some embodiments, the CD-2 binding molecules provided herein decrease the number of CD2hlghTmem cells, e.g., central memory (Tcm) and effector memory (Tem) T cells, while preserving regulatory T naive cells. In some embodiments, decreasing the number of CD2hlghTmem cells, e.g., central memory (Tcm) and effector memory (Tem) T cells, while preserving regulatory T naive cells, in the individual preconditions the individual for subsequent administration of the composition comprising the modified Treg cells. In some embodiments, the CD-2 binding molecules provided herein increase the Treg / Temratio or the Treg / Tcmratio, e.g., in CD4+and / or CD8+T cells. In some embodiments, increasing the Treg / Tem ratio or the Treg / Tcmratio, e.g., in CD4+and / or CD8+T cells, in the individual preconditions the individual for subsequent administration of the composition comprising the modified Treg cells. In some embodiments, the CD-2 binding molecules provided herein increase the proportion of CD4+Temcells expressing PD-1 and / or TIGIT. In some embodiments, increasing the proportion of CD4+Temcells expressing PD-1 and / or TIGIT in the individual preconditions the individual for subsequent administration of the composition comprising the modified Treg cells. In some embodiments, preconditioning the individual by administering to the individual the CD-2 binding molecule prior to administration of the composition comprising the modified Treg cells improves the efficacy of the modified Treg cells. In some embodiments, the efficacy of the modified Treg cells after preconditioning is improved as compared to the efficacy of the modified Treg cells being administered as a monotherapy.11MOFO-360045222Docket No.: 237752001540

[0047] Without wishing to be bound by theory, the CD-2 binding molecules provided herein bind to CD2, a cell surface protein expressed most prominently on CD4+and CD8+Tem (T effector memory) cells, the cells primarily responsible for beta cell destruction in type 1 Diabetes (T1D). The efficacy of the CD-2 binding molecules in the treatment of autoimmune disease is partially due to its ability to increase the relative amounts of Treg cells versus effector T cells, and thus, its efficacy can be further enhanced by subsequent administration of compositions comprising modified Treg cells. Accordingly, administration of the combination therapy provided herein may lead to extended preservation of endogenous insulin production, reduction in insulin requirements, decrease in the rate of major hypoglycemia, and restoration of beta cells in T1D patients. In some embodiments, administration of the combination therapy provided herein can be used for the treatment of an autoimmune disease or inflammatory disease, such as type I diabetes, rheumatoid arthritis, hidradenitis suppurativa, amyotrophic lateral sclerosis, celiac disease, and inflammatory bowel disease.I. Dose Schedule

[0048] Presented herein are methods of treating an autoimmune disease or inflammatory disease in an individual, comprising administering to the individual: a) about 1 mg to about 30 mg of a CD-2 binding molecule comprising a CD2-binding domain of human leukocyte function-associated antigen 3 (LFA3) linked to an Fc domain; and b) a composition comprising modified regulatory T (Treg) cells, wherein the modified Treg cells express a therapeutic protein; and wherein the composition comprising the modified Treg cells is administered after administration of the CD-2 binding molecule. In some embodiments, administration of the CD-2 binding molecule results in a reduction in the levels of CD2 positive cells in the individual, thus preconditioning the individual for treatment with the composition comprising the modified Treg cells.

[0049] In some embodiments, administration of the composition comprising the modified Treg cells is delayed such that depletion of the modified Treg cells by the CD-2 binding molecule is minimized. In some embodiments, the composition comprising the modified Treg cells is administered at least about 12 days, at least about 14 days, at least about 15 days, at least about 21 days, at least about 28 days, at least about 30 days, at least about 35 days, at least about 42 days, at least about 50 days, at least about 60 days, at least about 70 days, at least about 80 days, at least about 90 days, at least about 100 days, at least about 110 days, at12MOFO-360045222Docket No.: 237752001540 least about 120 days, at least about 140 days, or at least about 160 days after administration of the CD-2 binding molecule. In some embodiments, the composition comprising the modified Treg cells is administered about 20 days to about 140 days after administration of the CD-2 binding molecule. In some embodiments, the composition comprising the modified Treg cells is administered about 50 days to about 120 days after administration of the CD-2 binding molecule. In some embodiments, the composition comprising the modified Treg cells is administered about 60 days to about 100 days after administration of the CD-2 binding molecule. In some embodiments, the composition comprising the modified Treg cells is administered about 80 days to about 90 days after administration of the CD-2 binding molecule.

[0050] In some embodiments, the method further comprises measuring the level of CD2 positive cells in the individual to determine when to administer the composition comprising the modified Treg cells following administration of the CD-2 binding molecule. Any suitable assay may be used to detect, enumerate, determine phenotype, assess function and / or monitor CD2 positive cells, and include, but are not limited to ELISPOT, ELISA, flow cytometry, assays assessing binding to the antigen or ligand, or any other suitable assay. In some embodiments, the levels of CD2 positive cells in the individual are measured before and after the individual has received or one more doses of the CD-2 binding molecule. In some embodiments, the CD2 positive cells are effector memory T cells (Tem). In some embodiments, the levels of CD4+Temcells in the individual are measured. In some embodiments, the levels of CD2 positive cells (e.g., CD4+Temcells) in the individual are not measured. In some embodiments, the composition comprising the modified Treg cells is administered when the level of CD4+ Temin the peripheral blood of the individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, compared to the level of CD4+ Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule. In some embodiments, the composition comprising the modified Treg cells is administered when the level of CD4+ Temin the peripheral blood of the individual is reduced by at least about 40% compared to the level of CD4+ Tem in the peripheral blood of the individual prior to administration of the CD-2 binding molecule. In some embodiments, the composition comprising the modified Treg cells13MOFO-360045222Docket No.: 237752001540 is administered when the level of CD4+ Temin the peripheral blood of the individual is reduced by about 10% to about 100%, about 20% to about 80%, or about 30% to about 60% compared to the level of CD4+ Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule. In some embodiments, the composition comprising the modified Treg cells is administered when the level of CD4+ Temin the peripheral blood of the individual is reduced by about 25% to about 50% compared to the level of CD4+ Temin the peripheral blood of the individual prior to administration of the CD- 2 binding molecule.

[0051] In some embodiments, the method further comprises measuring the level of CD2 positive cells in the individual to determine when to administer the composition comprising the modified Treg cells. In some embodiments, the levels of CD2 positive cells in the individual are measured before and after the individual has received or one more doses of the CD-2 binding molecule. In some embodiments, the CD2 positive cells are central memory T cells (Tcmcells). In some embodiments, the levels of CD4 positive Tcmcells in the individual are measured. In some embodiments, the levels of CD2 positive cells (e.g., CD4 positive Tcmcells) in the individual are not measured. In some embodiments, the composition comprising the modified Treg cells is administered when the level of CD4+ Tcmin the peripheral blood of the individual is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% compared to the level of CD4+ Tcmin the peripheral blood of the individual prior to administration of the CD-2 binding molecule. In some embodiments, the composition comprising the modified Treg cells is administered when the level of CD4+ Tcmin the peripheral blood of the individual is reduced by at least about 40% compared to the level of CD4+ Tcmin the peripheral blood of the individual prior to administration of the CD- 2 binding molecule. In some embodiments, the composition comprising the modified Treg cells is administered when the level of CD4+ Tcmin the peripheral blood of the individual is reduced by about 10% to about 100%, about 20% to about 80%, or about 30% to about 60% compared to the level of CD4+ Tcmin the peripheral blood of the individual prior to administration of the CD-2 binding molecule. In some embodiments, the composition comprising the modified Treg cells is administered when the level of CD4+ Tcmin the peripheral blood of the individual is reduced by about 30% to about 50% compared to the14MOFO-360045222Docket No.: 237752001540 level of CD4+ Tcmin the peripheral blood of the individual prior to administration of the CD- 2 binding molecule.

[0052] In some embodiments, the method further comprises measuring the concentration of the CD-2 binding molecule in a sample of blood, serum, or plasma from the individual to determine when to administer the composition comprising modified Treg cells. In some embodiments, administration of the composition comprising the modified Treg cells is delayed such that depletion of the modified Treg cells by the CD-2 binding molecule is minimized. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is less than about 100 ng / mL, less than about 90 ng / mL, less than about 80 ng / mL, less than about 70 ng / mL, less than about 60 ng / mL, less than about 50 ng / mL, less than about 40 ng / mL, less than about 30 ng / mL, less than about 20 ng / mL, less than about 10 ng / mL, or less than about 5 ng / mL. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is less than about 50 ng / mL. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is less than about 20 ng / mL. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is about 5 ng / mL to about 100 ng / mL, about 12 ng / mL to about 80 ng / mL, about 15 ng / mL to about 60 ng / mL, about 18 ng / mL to about 50 ng / mL, or about 20 ng / mL to about 40 ng / mL. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is about 10 ng / mL to about 50 ng / mL.

[0053] In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is less than about 50 ng / mL, wherein the dose of the CD-2 binding molecule is about 3 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is less than about 50 ng / mL, wherein the dose of the CD-2 binding molecule is about 5 mg, and wherein the CD-2 binding15MOFO-360045222Docket No.: 237752001540 molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is less than about 50 ng / mL, wherein the dose of the CD-2 binding molecule is about 7 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is less than about 50 ng / mL, wherein the dose of the CD-2 binding molecule is about 10 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is less than about 50 ng / mL, wherein the dose of the CD-2 binding molecule is about 3 mg, and wherein the CD-2 binding molecule is administered intravenously.

[0054] In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is less than about 20 ng / mL, wherein the dose of the CD-2 binding molecule is about 3 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is less than about 20 ng / mL, wherein the dose of the CD-2 binding molecule is about 5 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is less than about 20 ng / mL, wherein the dose of the CD-2 binding molecule is about 7 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is less than about 20 ng / mL, wherein the dose of the CD-2 binding molecule is about 10 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from16MOFO-360045222Docket No.: 237752001540 the individual is less than about 20 ng / mL, wherein the dose of the CD-2 binding molecule is about 3 mg, and wherein the CD-2 binding molecule is administered intravenously.

[0055] In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is about 10 ng / mL to about 50 ng / mL, wherein the dose of the CD-2 binding molecule is about 3 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is about 10 ng / mL to about 50 ng / mL, wherein the dose of the CD-2 binding molecule is about 5 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is about 10 ng / mL to about 50 ng / mL, wherein the dose of the CD-2 binding molecule is about 7 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is about 10 ng / mL to about 50 ng / mL, wherein the dose of the CD-2 binding molecule is about 10 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the sample of blood, serum, or plasma from the individual is about 10 ng / mL to about 50 ng / mL, wherein the dose of the CD-2 binding molecule is about 3 mg, and wherein the CD-2 binding molecule is administered intravenously.

[0056] In some embodiments, administration of the composition comprising modified Treg cells is determined by the dosage of the CD-2 binding molecule. In some embodiments, when a higher dosage of CD-2 binding molecule is administered, the composition comprising the modified Treg cells is administered later than when a lower dosage of the CD-2 binding molecule is administered. In some embodiments, the composition comprising the modified Treg cells is administered at least about 12 days, at least about 14 days, at least about 15 days, at least about 21 days, at least about 28 days, at least about 30 days, at least about 35 days, at least about 42 days, at least about 50 days, at least about 60 days, at least about 70 days, at least about 80 days, at least about 90 days, at least about 100 days, at least about 11017MOFO-360045222Docket No.: 237752001540 days, at least about 120 days, at least about 140 days, or at least about 160 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 1 mg to about 30 mg.

[0057] In some embodiments, administration of the composition comprising modified Treg cells is determined by the dosage and route of administration of the CD-2 binding molecule. In some embodiments, the composition comprising the modified Treg cells is administered about 65 days to about 80 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 3 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered about 75 days to about 95 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 5 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered about 80 days to about 105 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 7 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered about 85 days to about 120 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 10 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered about 55 days to about 80 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 3 mg, and wherein the CD-2 binding molecule is administered intravenously.

[0058] In some embodiments, the composition comprising the modified Treg cells is administered about 67 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 3 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered about 76 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 3 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered about 76 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 5 mg, and wherein the CD-2 binding molecule is administered by intramuscular18MOFO-360045222Docket No.: 237752001540 injection. In some embodiments, the composition comprising the modified Treg cells is administered about 93 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 5 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered about 83 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 7 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered about 103 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 7 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered about 89 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 10 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered about 118 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 10 mg, and wherein the CD-2 binding molecule is administered by intramuscular injection. In some embodiments, the composition comprising the modified Treg cells is administered about 56 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 3 mg, and wherein the CD-2 binding molecule is administered intravenously. In some embodiments, the composition comprising the modified Treg cells is administered about 77 days after administration of the CD-2 binding molecule, wherein the dose of the CD-2 binding molecule is about 3 mg, and wherein the CD-2 binding molecule is administered intravenously.II. Compositions Comprising Modified Treg Cells

[0059] Presented herein is are methods of treating an autoimmune disease or inflammatory disease in an individual, comprising administering to the individual: a) about 1 mg to about 30 mg of a CD-2 binding molecule comprising a CD2-binding domain of human leukocyte function-associated antigen 3 (LFA3) linked to an Fc domain; and b) a composition comprising modified regulatory T (Treg) cells, wherein the modified Treg cells express a therapeutic protein; and wherein the composition comprising the modified Treg cells is administered after administration of the CD-2 binding molecule.19MOFO-360045222Docket No.: 237752001540

[0060] “Regulatory T cells,” or “Tregcells,” are cells belonging to a specialized subpopulation of T cells that act to suppress immune response, thereby maintaining homeostasis and self-tolerance. Tregcells are able to inhibit T cell proliferation and cytokine production and play a critical role in preventing autoimmunity. Tregcells are characterized by expression of FoxP3. Surface markers for Tregcells include CD4, CD25high (high molecular density) and CD1271ow (low molecular density). Mouse and human Treg cells express GITR / AITR, and CTLA-4. Human CD4+FoxP3+ cells can be divided into three sub-populations: (1) CD45RA+CD25+FoxP31ow resting Treg cells, (2) CD45RO+CD25highFoxP3high activated Treg cells, and (3) proinflammatory cytokine-producing CD45RO+CD25+FoxP31ow nonsuppressive T cells. In some embodiments, the modified Treg cells are CD4+, CD25+ and CD1271o.

[0061] In some embodiments, a Treg cell comprises a nucleic acid molecule encoding a therapeutic protein, wherein the nucleic acid molecule further comprises an expression control sequence operatively linked with the nucleotide sequence encoding the therapeutic protein. The nucleic acid molecule encoding the therapeutic protein can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the therapeutic protein in the Treg cell. Correct assembly can be confirmed by nucleotide sequencing, restriction mapping, and / or expression of the therapeutic protein in a Treg cell or any other suitable host. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.

[0062] In some embodiments, the modified Treg cells express a therapeutic protein comprising an antigen binding domain. In some embodiments, the therapeutic protein is an antibody, an engineered antibody such as an scFv, a CAR, an engineered TCR, a TCR mimic or a chimeric antibody-T cell receptor, or a chimeric signaling receptor. In some embodiments, the therapeutic protein of the modified Treg cells is a T cell receptor (TCR) comprising an antigen-binding site that specifically binds to antigen. In some embodiments, the therapeutic protein of the modified Treg cells is a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain that specifically binds to an antigen. In some embodiments, the antigen is associated with an autoimmune disease or inflammatory disease. In some embodiments, the antigen is selected from MHC class I, MHC class II, collagen type V, K alphal tubulin, MHC class I related chain A (MICA), fibronectin, collagen20MOFO-360045222Docket No.: 237752001540 type IV, collagen type VI, vimentin, angiotensin II type 1 receptor (AGTR1), perlecan, agrin, collagen type I, collagen type II, collagen type III, cardiac myosin, an islet cell autoantibody (ICA) antigen, insulin, glutamic acid decarboxylase (GAD), myelin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, astrocyte proteins, glial fibrillary protein (GFAP), SlOObeta, beta cell antigen, insulin B chain, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD65), islet-associated antigen 2, islet-specific glucose-6- phosphatase catalytic subunit-related protein (IGRP), zinc transporter 8 (ZnT8), islet antigen 2 (IA-2), heat shock protein 60 (HSP60), chromogranin A, dnaJ (heat shock protein), citrullinated-vimentin, human cartilage glycoprotein-39, mitochondrial component, pyruvate dehydrogenase (mitochondrial), E2 component of pyruvate dehydrogenase, E2 component of branched chain 2-oxo acid dehydrogenase, E2 component of 2-oxo-glutarate dehydrogenase complex, E3 binding protein of dihydrolipoamide dehydrogenase, a nuclear component, nuclear protein splOO, nuclear pore complex protein gpl20, centromere, acetylcholine receptor (AChR), aquaporin-4 (AQP-4), CTLA-4, ICAM, LFA-3, CD40 / CD154, ICOS / ICOSL, CD52, nuclear factor of activated T cells (NF AT), phospholipase C (PLC), CD25, Janus kinase, B cell activating factor (BAFF), a proliferating inducing ligand (APRIL), IL6R, IL 17, IL12 / IL23, an integrin, a sphingosin receptor, melanocyte antigen, tolllike receptor, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, MyD88, an IL-lR-associated kinase (IRAK), an allergen associated with the allergic disorder, a citrullinated protein or citrullinated fragments thereof, citrullinated vimentin, and citrullinated filaggrin. In some embodiments, the antigen binding domain of the CAR specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof. In some embodiments, the antigen binding domain of the CAR specifically binds to citrullinated vimentin. In some embodiments, the antigen binding domain of the CAR binds to all three of (i) citrullinated vimentin, (ii) citrullinated filaggrin, and (iii) citrullinated fibrinogen, or citrullinated peptides fragments thereof, optionally wherein the antigen binding domains further binds to citrullinated tenascin C.

[0063] In some embodiments, the modified Treg cells express one or more additional therapeutic proteins.

[0064] In some embodiments, the composition comprising modified Treg cells comprises one or more types of modified Treg cells. For example, the composition comprises at least 2, at least 3, or at least 4 types of modified Treg cells, wherein each type of modified Treg cell expresses a different therapeutic protein.21MOFO-360045222Docket No.: 237752001540

[0065] In some embodiments, the modified Treg cells express a therapeutic protein, wherein the therapeutic protein is a CAR. “Chimeric antigen receptors” or “CARs” are engineered molecules comprising an optional signal peptide, a target binding domain, an optional hinge region, a transmembrane domain, an intracellular signaling domain and an optional co-stimulatory domain. CARs are based on the structure of T cell receptors, which are expressed on T cells and which are involved in the cell-mediated immune response. The “target binding domain” is also referred to herein as an “antigen binding domain”, and as such the term “target” encompasses an “antigen.”

[0066] So-called “first-generation” CARs had a targeting domain and a CD3^ signal transduction domain. So-called “second generation” CARs further included a co-stimulatory domain, such as a CD28 or 4- IBB domain. So-called “third generation” CARs comprise multiple co-stimulatory domains. So-called “fourth generation” CARs, also referred to as “TRUCKS” are engineered to release a transgenic cytokine upon CAR signaling.

[0067] Chimeric antigen receptors (“CARs”) may include the following elements: (1) an optional signal peptide, (2) a target binding domain, (3) an optional hinge region; (4) a transmembrane region; (5) an intracellular domain comprising a signal transduction domain. Optionally, the CAR includes any of a CD3(^ signal transduction domain, an Fc receptor signal transduction domain, a co-stimulatory (signal transduction) domain. That is, these optional elements are included in addition to or instead of other optional elements. The target binding domain is heterologous to at least one of the other domains. That is, the target binding domain does not naturally occur on a T cell receptor, or is not in the same protein as at least one of the other domains.

[0068] The “target binding domain” or “antigen binding domain” provides binding specificity to the CAR. The “signal peptide” guides the polypeptide through the cell membrane. The target binding domain can bind to a domain of an antibody that binds to the target antigen for a so-called “Universal CAR”. The “hinge region” is a flexible connector region, e.g., a natural or synthetic polypeptide, or any other type of molecule, providing structural flexibility and spacing to flanking polypeptide regions. The “transmembrane domain” is a membrane-spanning protein domain, typically hydrophobic. The “signal transduction domain” or “signaling domain” transmits a signal through a signal transduction pathway into the cell upon binding. Such signaling activates an activity of the cell. “Co- stimulatory domains” are accessory signaling domains that further transmit signals.22MOFO-360045222Docket No.: 237752001540

[0069] In some embodiments, the CAR comprises: (i) an target-binding domain (also referred to herein as an antigen binding domain) reactive with any antigen that is associated with an autoimmune disease or inflammatory disease, such as a VH-VL or VL-VH, wherein the two variable domains are separated by a flexible linker of from 15-25 amino acids in length; (ii) a hinge domain; (iii) a co-stimulatory domain; and (iv) an intracellular signaling domain (also referred to herein as an activation domain). That is, in some embodiments the CAR comprises an antigen-binding domain fused to a CAR platform comprising a hinge domain, a transmembrane domain, and an intracellular domain comprising a costimulatory domain and an activation domain. The CAR may further include a signal peptide (also referred to herein as a leader sequence) to direct expression of the CAR to the surface of a the Treg cell.

[0070] In some embodiments, the CAR comprises an intracellular signaling domain, wherein the intracellular signaling domain is derived from CD3-zeta. In some embodiments, the CAR comprises at least one co-stimulatory domain, wherein the at least one costimulatory domain a co-stimulatory domain of a member of the group consisting of FceRlg, Fcg, CD28, CD134 (0X40), CD137 (4-1BB), CTLA-4, CTLA-4 / CD-28 hybrid, DAP10, CD27, 2B4, and combinations thereof. In some embodiments, the CAR comprises an antigenbinding domain, wherein the antigen-binding domain comprises an antibody, an antibody fragment, a camelid nanobody, a heavy chain only antibody or an aptamer. In some embodiment, the antigen-binding domain is a single chain variable fragment (scFv). In some embodiments, the CAR comprises a transmembrane domain, wherein the transmembrane domain is a CD8 transmembrane domain or a CD28 transmembrane domain. In some embodiments, the CAR comprises a hinge domain, wherein the hinge domain is a CD8 hinge domain or a CD28 hinge domain. In some embodiments, the CAR further comprising a signal peptide. In some embodiments, the signal peptide of the CAR is a CD8 signal peptide or a GM-CSF signal peptide.

[0071] The Treg cells to be transformed with the nucleic acids encoding a therapeutic protein can be cells taken from a subject into whom the cells are to be administered. In this way, issues of an allogeneic immune response can be mitigated.

[0072] Cells can be expanded ex vivo before administration to a subject.23MOFO-360045222Docket No.: 237752001540

[0073] Treg cells into which nucleic acids expressing the therapeutic protein have been incorporated can express the therapeutic protein and be used in the methods described herein to treat autoimmune disease or inflammatory disease.

[0074] In some embodiments, the modified Treg cells are human T cells. In some embodiments, the modified Treg cells are a primary T cells.III. CD-2 Binding Molecules

[0075] Provided herein are methods of treating an autoimmune disease or inflammatory disease in an individual, comprising administering to the individual: a) about 1 mg to about 30 mg of a CD-2 binding molecule comprising a CD2-binding domain of human leukocyte function-associated antigen 3 (LFA3) linked to an Fc domain; and b) a composition comprising modified regulatory T (Treg) cells, wherein the modified Treg cells express a therapeutic protein; and wherein the composition comprising the modified Treg cells is administered after administration of the CD-2 binding molecule. In some embodiments the CD-2 binding molecule is an isolated CD-2 binding molecule. In some embodiments, an isolated CD-2 binding molecule is administered to the individual.

[0076] Such CD-2 binding molecules specifically bind to the transmembrane glycoprotein CD2. The CD-2 binding molecule is an immunomodulatory dimeric fusion protein comprising an engineered CD2-binding domain of human leukocyte function-associated antigen 3 (LFA-3) linked to an Fc portion of human immunoglobulin G1 (IgGl). In some embodiments, the CD-2 binding molecule is a modified version of the approved alefacept.

[0077] In some embodiments, the CD-2 binding molecules have been engineered to improve their stability and manufacturability (e.g., for improved production) relative to CD-2 binding molecules comprising the wild type LFA3 sequence (e.g., alefacept). In some embodiments, the CD-2 binding molecules have been engineered to increase their binding affinity to CD2 relative to CD-2 binding molecules comprising the wild type LFA3 sequence (e.g., alefacept). In some embodiments, the CD-2 binding molecules provided herein share the same mechanism of action as alefacept (AMEVIVE, 2011; Miller et al., 1993; Majeau et al., 1994) and have been specifically designed to optimize stability, pharmacokinetic (PK), and manufacturability and in turn its biological function in vivo.

[0078] In some embodiments, the CD-2 binding molecules provided herein have been engineered to have extended half-life. An improvement in the half-life can have many therapeutic advantages. For instance, an improved half-life can allow for less frequent dosing24MOFO-360045222Docket No.: 237752001540 to maintain the desired exposures and avoid unnecessarily high drug peak concentrations. Other advantages of the improved half-life include but are not limited to ease of achieving optimal efficacy, ease of determining the dosing frequency, safety and patient compliance.

[0079] In some embodiments, the CD-2 binding molecules of the present disclosure comprise the amino acid sequence of SEQ ID NO:2. In some embodiments, the CD-2 binding molecules of the present disclosure comprise an LFA3 domain and an Fc domain. In some embodiments, the LFA3 domain of the CD-2 binding molecules of the present disclosure comprises SEQ ID NO: 3, which contains four amino acid substitutions as compared to wildtype LFA3 domain. In some embodiments, the LFA3 domain comprises an amino acid sequence with at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the Fc domain of the CD-2 binding molecules of the present disclosure comprise SEQ ID NO: 4, which comprises the hinge, CH2 and CH3 domains of the human immunoglobulin 1 (IgGl). In some embodiments, the Fc domain comprises an amino acid sequence with at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD-2 binding molecules of the present disclosure is encoded by the nucleotide sequence of SEQ ID NO: 1, or may be encoded by any other nucleotide sequence determined by one of skill in the art to encode SEQ ID NO: 2. In some embodiments, the LFA3 domain of CD-2 binding molecules of the present disclosure (SEQ ID NO: 3) has been engineered to contain four amino acid substitutions in the LFA3 extracellular domain (A36V, L38F, F43 V, and M86F) to improve stability, production, and manufacturability. In some embodiments, the LFA3 domain of the CD-2 binding molecules of the present disclosure comprises four predicted N-linked glycosylation sites (N12, N66, N81, and N170). An exemplary CD-2 binding molecule of the present disclosure is LF-vl, which is the subject of the Examples herein. LF-vl is an immunomodulatory dimeric fusion protein comprising the engineered cluster of differentiation (CD) 2-binding domain of human leukocyte function antigen-3 (LFA-3) linked to the fragment crystallizable region (Fc) portion of human immunoglobulin G1 (IgGl). Without wishing to be bound by theory, LF-vl may reduce the number of memory CD4 and CD8 T cells through ADCC / cytotoxicity while relatively25MOFO-360045222Docket No.: 237752001540 sparing Tregand Tnaive cells, and may modulate CD2 / LFA3 interaction, leading to improvement in Treg / Temand Treg / TCM ratios in both CD4+ T cells and CD8+ T cells.

[0080] In some embodiments, the methods of treating an autoimmune disease or inflammatory disease in an individual comprises administering to the individual a functional variant of the CD-2 binding molecule. As used herein a “functional variant” of a CD-2 binding molecule is one that shares one or more biological functions with a CD-2 binding molecule comprising the amino acid sequence set forth in SEQ ID NO:2 and has one or more amino acid substitutions, insertions, and / or deletions. In some embodiments, a functional variant of the CD-2 binding molecule comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:2. In some embodiments, a functional variant of the CD-2 binding molecule comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:2 and comprises four amino acid substitutions (including A36V, L38F, F43V, and M86F) in the LFA3 domain. In some embodiments, a functional variant of the CD-2 binding molecule comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:2 and exhibits one or more of the following functions: (i) preferential binding to CD2hlghTemcells, e.g., CD4+and / or CD8+Temcells in vivo, (ii) killing CD2-expressing cells (e.g., CD4+or CD8+TCM cells, or CD4+or CD8+Temcells) in the presence of NK cells, (iii) decreasing CD4+and / or CD8+Temcells, e.g., peripheral CD4+Temcells, and (iv) increasing the Treg / Tem ratio, e.g., in CD4+T cells, and / or CD8+T cells. In some embodiments, a functional variant of the CD-2 binding molecule comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO:2, comprises four amino acid substitutions (including A36V, L38F, F43V, and M86F) in the LFA3 domain, and exhibits one or more of the following functions: (i) preferential binding to CD2hlghTemcells, e.g., CD4+and / or CD8+Tem cells in vivo, (ii) killing CD2-expressing cells (e.g., CD4+or CD8+TCM cells, or CD4+or CD8+Tem cells) in the presence of NK cells, (iii) decreasing CD4+and / or CD8+Temcells, e.g., peripheral CD4+Temcells, and (iv) increasing the Treg / Temratio, e.g., in CD4+T cells, and / or CD8+T cells.

[0081] In some embodiments, the CD-2 binding molecule provided herein comprises an engineered Fc polypeptide comprising a C- terminal lysine (K) amino acid residue (e.g., human IgGl heavy chain comprising a terminal lysine). One skilled in the art would understand that the lysine residue may be clipped resulting in a fusion protein lacking the C-26MOFO-360045222Docket No.: 237752001540 terminal lysine residue. Thus, in some embodiments, the CD-2 binding molecule provided herein comprises a polypeptide where the terminal lysine is not present.

[0082] In some embodiments, the CD-2 binding molecule is a multimeric (e.g., dimeric) polypeptide molecule. In some embodiments, the CD-2 binding molecule comprises two or more polypeptide molecules comprising the amino acid sequence of SEQ ID NO: 2.

[0083] In some embodiments, the CD-2 binding molecule modulates the function of and selective depletion of CD2+Temcells to rebalance the regulatory / Temcell numbers. In some embodiments, the CD-2 binding molecule promotes elimination and / or suppression of pathogenic T effector cells. In some embodiments, the CD-2 binding molecule modulates the interaction between CD2 and LFA3, thereby interrupting CD2-mediated T cell costimulation.

[0084] In some embodiments, the CD-2 binding molecule depletes CD2+T cells via FcR- mediated antibody dependent cellular cytotoxicity (ADCC). In some embodiments, the CD-2 binding molecule depletes CD2+T cells via apoptosis. In some embodiments, the CD-2 binding molecule decreases the number of CD2hlghT memory cells (Tmem), e.g., central memory (Tcm) and effector memory (Tem) T cells, while preserving regulatory T cells (Treg). In some embodiments, the CD-2 binding molecule decreases the number of CD2hlghTmem cells, e.g., central memory (Tcm) and effector memory (Tem) T cells, while preserving regulatory T naive cells. In some embodiments, the CD-2 binding molecule increases the Treg / Tem ratio or the Treg / Tcmratio, e.g., in CD4+and / or CD8+T cells. In some embodiments, the CD-2 binding molecule increases the proportion of CD4+Temcells expressing PD-1 and / or TIGIT.

[0085] In some embodiments, the CD-2 binding molecule binds to CD2, a cell surface protein expressed most prominently on T effector memory (CD4+Temand CD8+Tem) cells. The efficacy of the CD-2 binding molecules in the treatment of autoimmune disease or inflammatory disease is partially due to its ability to increase the relative amounts of Treg cells versus effector T cells, and thus, its efficacy can be further enhanced by subsequent administration of compositions comprising Treg cell therapies. Accordingly, administration of the of the combination therapy provided herein may be used to treat an autoimmune disease or inflammatory disease such as type I diabetes, rheumatoid arthritis, hidradenitis suppurativa, amyotrophic lateral sclerosis, celiac disease, and inflammatory bowel disease.27MOFO-360045222Docket No.: 237752001540

[0086] In some embodiments, the CD-2 binding molecule binds to CD2-expressing cells, e.g., CD4+Tmem cells, with a calculated Kd that is no more than about 100, 200, 300, 400 or 500 pM. In some embodiments, the CD-2 binding molecule binds to CD4+Tcmcells, with a calculated Kd that is no more than about 100, 200, 300, 400, or 500pM. In some embodiments, the CD-2 binding molecule binds to CD4+naive T cells, with a calculated Kd that is no more than about 100, 200, 300 or 400 pM. In some embodiments, the CD-2 binding molecule binds to Tregcells, with a calculated Kd that is no more than about 100, 200 or 300 pM. In some embodiments, the CD-2 binding molecule binds to CD8+memory T cells, with a calculated Kd that is no more than about 50, 100 or 150 pM. In some embodiments, the CD-2 binding molecule binds to CD8+naive T cells, with a calculated Kd that is no more than about 50, 100, 200, 300, 400, or 500pM.

[0087] In some embodiments, the CD-2 binding molecule binds to CD4+memory T cells with a calculated IC50 that is no more than about 300, 400, 500, 700, 800, 1000, 1200 or 1500 pM. In some embodiments, the CD-2 binding molecule binds to CD4+Temthat is no more than about 150, 300, 400, 500, 600, 700, 800, 1000 or 1200 pM. In some embodiments, the CD-2 binding molecule binds to CD4+Tcmcells with a calculated IC50 that is no more than about 100, 200, 300, 400, 500, 600, 700 or 800 pM. In some embodiments, the CD-2 binding molecule binds to CD4+naive T cells with a calculated IC50 that is no more than about 200, 300, 400, 500, 600, 700, 800, 1000 or 1200 pM. In some embodiments, the CD-2 binding molecule binds to Tregcells with a calculated IC50 that is no more than about 100, 200, 300, 400 or 500 pM. In some embodiments, the CD-2 binding molecule binds to CD8 memory T cells with a calculated IC50 that is no more than about 100, 200, 300, 400, 500 or 600 pM. In some embodiments, the CD-2 binding molecule binds to CD8+naive T cells with a calculated IC50 that is no more than about 300, 400, 500, 600, 700, 800, 1000, 1200, 1500, 1600 or 1700 pM.A. Sequences28MOFO-360045222Docket No.: 23775200154029MOFO-360045222Docket No.: 237752001540B. Pharmaceutical Dosages

[0088] The present disclosure provides methods of treating an autoimmune disease or inflammatory disease comprising administering to an individual a combination therapy comprising: a) a CD-2 binding molecule comprising a CD2-binding domain of human leukocyte function-associated antigen 3 (LFA3) linked to an Fc domain and b) a composition comprising modified regulatory T (Treg) cells, wherein the modified Treg cells express a therapeutic protein. In some embodiments, the CD-2 binding molecule comprises the sequence of amino acids set forth in SEQ ID NO: 2 or a functional variant thereof. In some embodiments, the CD-2 binding molecule is administered to the individual at a dose of about 1 mg to about 30 mg. In some embodiments, the dose of the CD-2 binding molecule is about 1 mg, about 3 mg, about 5 mg, about 7 mg, about 10 mg, or about 30 mg. In some embodiments, the dose of the CD-2 binding molecule is about 1 mg. In some embodiments, the dose of the CD-2 binding molecule is about 2 mg. In some embodiments, the dose of the CD-2 binding molecule is about 3 mg. In some embodiments, the dose of the CD-2 binding molecule is about 4 mg. In some embodiments, the dose of the CD-2 binding molecule is about 5 mg. In some embodiments, the dose of the CD-2 binding molecule is about 6 mg. In some embodiments, the dose of the CD-2 binding molecule is about 7 mg. In some embodiments, the dose of the CD-2 binding molecule is about 8 mg. In some embodiments, the dose of the CD-2 binding molecule is about 9 mg. In some embodiments, the dose of the CD-2 binding molecule is about 10 mg. In some embodiments, the dose of the CD-2 binding molecule is about 11 mg. In some embodiments, the dose of the CD-2 binding molecule is about 12 mg. In some embodiments, the dose of the CD-2 binding molecule is about 13 mg. In some embodiments, the dose of the CD-2 binding molecule is about 14 mg. In some embodiments, the dose of the CD-2 binding molecule is about 15 mg. In some embodiments, the dose of the CD-2 binding molecule is about 16 mg. In some embodiments, the dose of the CD-2 binding molecule is about 17 mg. In some embodiments, the dose of the CD-2 binding molecule is about 18 mg. In some embodiments, the dose of the CD-2 binding molecule is about 19 mg. In some embodiments, the dose of the CD-2 binding molecule is about 20 mg. In some embodiments, the dose of the CD-2 binding molecule is about 21 mg. In some embodiments, the dose of the CD-2 binding molecule is about 22 mg. In some embodiments, the dose of the CD-2 binding molecule is about 23 mg. In some embodiments, the dose of the CD-2 binding molecule is about 24 mg. In some embodiments, the dose of the CD-2 binding molecule is about 25 mg. In some embodiments, the dose of the CD-2 binding molecule is30MOFO-360045222Docket No.: 237752001540 about 26 mg. In some embodiments, the dose of the CD-2 binding molecule is about 27 mg. In some embodiments, the dose of the CD-2 binding molecule is about 28 mg. In some embodiments, the dose of the CD-2 binding molecule is about 29 mg. In some embodiments, the dose of the CD-2 binding molecule is about 30 mg.

[0089] In some embodiments, the CD-2 binding molecule provided herein is administered at a dose of about 1 mg to about 30 mg per administration (e.g., injection). For instance, the CD-2 binding molecule provided herein is administered intramuscularly at a dose of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, or about 30 mg per administration (e.g., injection). In some embodiments, about 3 mg, about 5 mg, about 7 mg, or about 10 mg of the CD-2 binding molecule provided herein is administered to the individual, wherein the CD-2 binding molecule is administered by intramuscular injection. Any suitable method for intramuscular injection can be implemented for administration of the CD-2 binding molecule provided herein. In some embodiments, intramuscular injection comprises injection of the CD-2 binding molecule using a needle and syringe into the muscle tissue of the individual. Exemplary injection sites include, but are not limited to, the deltoid muscle of the arm, vastus lateralis muscle of the thigh, ventrogluteal muscle of the hip, or dorsogluteal muscles of the buttocks.

[0090] In some embodiments, about 3 mg of the CD-2 binding molecule provided herein is administered to the individual, wherein the CD-2 binding molecule is administered intravenously. Any suitable method for intravenous administration can be implemented for administration of the CD-2 binding molecule provided herein. In some embodiments, intravenous administration comprises injection of the CD-2 binding molecule provided herein using a needle or catheter directly into the vein of the individual. Exemplary injection sites include, but are not limited to, the vein of the wrist, elbow, or back of the hand. In some embodiments, the CD-2 binding molecule provided herein is administered through an intravenous “push” or “bolus” injection, wherein the CD-2 binding molecule is injected into the vein rapidly. In some embodiments, the CD-2 binding molecule provided herein is administered through an intravenous infusion (e.g., pump or drip infusion), wherein the CD-2 binding molecule is administered over time.31MOFO-360045222Docket No.: 237752001540

[0091] In some embodiments, the half-life (ti / 2) of the CD-2 binding molecule is the length of time required for the concentration of CD-2 binding molecules of the present disclosure to decrease to half of its starting dose in the body (e.g., in the blood, serum, or plasma) after administration to the individual. In some embodiments, the half-life is a linear half-life (ti / 2 linear) and is calculated as ln(2) / terminal rate constant (Xzlinear). In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding provided herein in the serum is about 10 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 11 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 12 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 13 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 14 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 15 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 16 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 17 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 18 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 19 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 20 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 21 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 22 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 23 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 24 days. In some embodiments, the linear half-life (ti / 2 linear) of a CD-2 binding molecule provided herein in the serum is about 25 days.

[0092] In some embodiments, the dose of the CD-2 binding molecule is about 1 mg and administered by intramuscular injection, wherein the linear half-life (ti / 2 linear) of the CD-2 binding molecule is about 5 days to about 20 days, about 8 days to about 16 days, or about 1032MOFO-360045222Docket No.: 237752001540 days to about 14 days. In some embodiments, the dose of the CD-2 binding molecule is about 3 mg and administered by intramuscular injection, wherein the linear half-life (ti / 2 linear) of the CD-2 binding molecule is about 10 days to about 30 days, about 15 days to about 25 days, or about 16 days to about 19 days. In some embodiments, the dose of the CD-2 binding molecule is about 3 mg and administered intravenously, wherein the linear half-life (ti / 2 linear) of the CD-2 binding molecule is about 5 days to about 25 days, about 10 days to about 20 days, or about 13 days to about 17 days. In some embodiments, the dose of the CD-2 binding molecule is about 10 mg and administered by intramuscular injection, wherein the linear halflife (ti / 2 linear) of the CD-2 binding molecule is about 10 days to about 30 days, about 15 days to about 25 days, or about 20 days to about 23 days. In some embodiments, the dose of the CD-2 binding molecule is about 30 mg and administered by intramuscular injection, wherein the linear half-life (ti / 2 linear) of the CD-2 binding molecule is about 5 days to about 25 days, about 10 days to about 20 days, or about 13 days to about 17 days.

[0093] In some embodiments, the CD-2 binding molecules provided herein exhibit an improved (e.g., longer or extended) half-life (ti / 2) and higher overall exposure as compared to alefacept. For instance, LF-vl, an exemplary CD-2 binding molecule of the present disclosure exhibits a longer half-life than alefacept, which has a half-life of 11.25 days for a 7.5 mg intravenous dose. An improvement in the half-life can have many therapeutic advantages. For instance, an improved half-life can allow for less frequent dosing to maintain the desired exposures and avoid unnecessarily high drug peak concentrations. Other advantages of the improved half-life include but are not limited to ease of achieving optimal efficacy, ease of determining the dosing frequency, safety, patient compliance.

[0094] The half-life of the CD-2 binding molecule provided herein in the blood (e.g., serum) of an individual is determined using any method known in the art, such as immunoassays, immunoblots, and mass spectrometry. In certain embodiments, the half-life of the CD-2 binding molecule of the disclosure in the blood (e.g., serum) of an individual is determined using an ELISA assay.

[0095] The C max comprises the highest (e.g., peak) concentration of a CD-2 binding molecule provided herein in the blood, serum, cerebrospinal fluid, or target organ after a dose has been administered. Drug quality bioavailability and bioequivalence rely on pharmacokinetic measurements such as Cmax andtmax, to assess extent of systemic absorption and area under the curve (AUC) to assess extent of systemic exposure. In some embodiments, the CD-2 binding molecule comprises the sequences of amino acids set forth in SEQ ID NO:33MOFO-360045222Docket No.: 2377520015402. In some embodiments, the maximum concentration (Cmax) of the CD-2 binding molecule in the serum of the individual is at least about 50 ng / mL, at least about 100 ng / mL, at least about 200 ng / mL, at least about 300 ng / mL, at least about 400 ng / mL, at least about 500 ng / mL, at least about 600 ng / mL, at least about 700 ng / mL, at least about 800 ng / mL, at least about 900 ng / mL, at least about 1,000 ng / mL, at least about 1,250 ng / mL, at least about 1,500 ng / mL, at least about 1,750 ng / mL, at least about 2,000 ng / mL, at least about 2,250 ng / mL, or at least about 2,500 ng / mL. In some embodiments, the maximum concentration (Cmax) of the CD-2 binding molecule in the serum of the individual is about 50 ng / mL to about 150 ng / mL, about 100 ng / mL to about 200 ng / mL, about 150 ng / mL to about 300 ng / mL, about 250 ng / mL to about 500 ng / mL, about 400 ng / mL to about 700 ng / mL, about 600 ng / mL to about 900 ng / mL, about 800 ng / mL to about 1,200 ng / mL, about 1,000 ng / mL to about 1,500 ng / mL, about 1,250 ng / mL to about 1,750 ng / mL, about 1,500 ng / mL to about 2,000 ng / mL, about 2,000 ng / mL to about 2,500 ng / mL, or about 2,250 ng / mL to about 3,000 ng / mL.

[0096] In some embodiments, the dose of the CD-2 binding molecule is about 1 mg and administered by intramuscular injection, wherein the maximum concentration (Cmax) of the CD-2 binding molecule in the serum of the individual is about 50 ng / mL to about 300 ng / mL, about 100 ng / mL to about 150 ng / mL, or about 100 ng / mL to about 125 ng / mL. In some embodiments, the dose of the CD-2 binding molecule is about 3 mg and administered by intramuscular injection, wherein the maximum concentration (Cmax) of the CD-2 binding molecule in the serum of the individual is about 200 ng / mL to about 500 ng / mL, about 300 ng / mL to about 400 ng / mL, or about 325 ng / mL to about 350 ng / mL. In some embodiments, the dose of the CD-2 binding molecule is about 3 mg and administered intravenously, wherein the maximum concentration (Cmax) of the CD-2 binding molecule in the serum of the individual is about 700 ng / mL to about 1,100 ng / mL, about 800 ng / mL to about 1,000 ng / mL, or about 900 ng / mL to about 950 ng / mL. In some embodiments, the dose of the CD-2 binding molecule is about 10 mg and administered by intramuscular injection, wherein the maximum concentration (Cmax) of the CD-2 binding molecule in the serum of the individual is about 800 ng / mL to about 1,200 ng / mL, about 900 ng / mL to about 1,100 ng / mL, or about 950 ng / mL to about 1,000 ng / mL. In some embodiments, the dose of the CD-2 binding molecule is about 30 mg and administered by intramuscular injection, wherein the maximum concentration (Cmax) of the CD-2 binding molecule in the serum of the individual is about 2,250 ng / mL to about 2,750 ng / mL, about 2,300 ng / mL to about 2,600 ng / mL, or about 2,400 ng / mL to about 2,500 ng / mL..34MOFO-360045222Docket No.: 237752001540

[0097] In some embodiments, the maximum concentration (Cmax) is a mean value e.g., geometric mean). In some embodiments, the dose of the CD-2 binding molecule is about 1 mg and administered by intramuscular injection, wherein the mean maximum concentration (Cmax) of the CD-2 binding molecule in the serum of the individual is about 113 ng / mL. In some embodiments, the dose of the CD-2 binding molecule is about 3 mg and administered by intramuscular injection, wherein the mean maximum concentration (Cmax) of the CD-2 binding molecule in the serum of the individual is about 340 ng / mL. In some embodiments, the dose of the CD-2 binding molecule is about 3 mg and administered intravenously, wherein the mean maximum concentration (Cmax) of the CD-2 binding molecule in the serum of the individual is about 918 ng / mL. In some embodiments, the dose of the CD-2 binding molecule is about 10 mg and administered by intramuscular injection, wherein the mean maximum concentration (Cmax) of the CD-2 binding molecule in the serum of the individual is about 995 ng / mL. In some embodiments, the dose of the CD-2 binding molecule is about 30 mg and administered by intramuscular injection, wherein the mean maximum concentration (Cmax) of the CD-2 binding molecule in the serum of the individual is about 2,480 ng / mL.

[0098] The maximum concentration (Cmax) of the CD-2 binding molecule of the disclosure in the blood (e.g., serum) of an individual is determined using any method known in the art, such as immunoassays, immunoblots, and mass spectrometry. In certain embodiments, the half-life of the CD-2 binding molecule of the disclosure in the blood (e.g., serum) of an individual is determined using an ELISA assay.

[0099] Another parameter to assess extent of systemic absorption (e.g., rate of systemic absorption) is the time to reach peak drug concentration or time of maximum observed concentration (tmax). In some embodiments, the tmax is less than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 hours. In some embodiments, the tmax is about 24 hours to about 48 hours. In some embodiments, the tmax is about 36 hours to about 72 hours. In some embodiments, the tmax is about 48 hours to about 72 hours. In some embodiments, the tmax is about 48 hours to about 96 hours. In some embodiments, the tmax is about 72 hours to about 120 hours. In some embodiments, the CD-2 binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the tmax of the CD-2 binding molecule is about 48 hours to about 94 hours. In some embodiments, the time to maximum concentration (tmax) of the CD-2 binding molecule is about 72 hours to about 94 hours.35MOFO-360045222Docket No.: 237752001540

[0100] The area under the curve (AUC) comprises a definite integral of the concentration of a drug in blood (e.g., plasma) as a function of time. The AUC from time zero to a given point in time (AUCt, wherein t specifies the time period over which AUC is calculated) is often measured to assess the extent of systemic exposure of a drug. In some embodiments, the CD- 2 binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the dose of the CD-2 binding molecule is about 1 mg and administered by intramuscular injection, wherein the area under the curve (AUCt) of the CD-2 binding molecule in the serum of the individual is about 0.50 pg*day / mL to about 4.0 pg*day / mL, about 1.0 pg*day / mL to about 3.0 pg*day / mL, or about 1.5 pg*day / mL to about 2.5 pg*day / mL. In some embodiments, the dose of the CD-2 binding molecule is about 3 mg and administered by intramuscular injection, wherein the area under the curve (AUCt) of the CD- 2 binding molecule in the serum of the individual is about 5.0 pg*day / mL to about 10 pg*day / mL, about 6.0 pg*day / mL to about 9.0 pg*day / mL, or about 7.0 pg*day / mL to about 8.0 pg*day / mL. In some embodiments, the dose of the CD-2 binding molecule is about 3 mg and administered intravenously, wherein the area under the curve (AUCt) of the CD-2 binding molecule in the serum of the individual is about 4.0 pg*day / mL to about 9.0 pg*day / mL, about 5.0 pg*day / mL to about 8.0 pg*day / mL, or about 6.0 pg*day / mL to about 7.0 pg*day / mL. In some embodiments, the dose of the CD-2 binding molecule is about 10 mg and administered by intramuscular injection, wherein the area under the curve (AUCt) of the CD-2 binding molecule in the serum of the individual is about 15 pg*day / mL to about 35 pg*day / mL, about 20 pg*day / mL to about 30 pg*day / mL, or about 20 pg*day / mL to about 25 pg*day / mL. In some embodiments, the dose of the CD-2 binding molecule is about 30 mg and administered by intramuscular injection, wherein the area under the curve (AUCt) of the CD-2 binding molecule in the serum of the individual is about 40 pg*day / mL to about 70 pg*day / mL, about 45 pg*day / mL to about 65 pg*day / mL, about 50 pg*day / mL to about 60 pg*day / mL, about 50 pg*day / mL to about 55 pg*day / mL, or about 55 pg*day / mL to about 60 pg*day / mL.

[0101] In some embodiments, AUC is measured from time zero to the time at which the last quantifiable concentration of the CD-2 binding molecule is observed (AUCiast). In some embodiments, the area under the curve (AUCiast) is a mean value (e.g., geometric mean). In some embodiments, the dose of the CD-2 binding molecule is about 1 mg and administered by intramuscular injection, wherein the mean area under the curve (AUCiast) of the CD-2 binding molecule in the serum of the individual is about 1.8 pg*day / mL. In some36MOFO-360045222Docket No.: 237752001540 embodiments, the dose of the CD-2 binding molecule is about 3 mg and administered by intramuscular injection, wherein the mean area under the curve (AUCiast) of the CD-2 binding molecule in the serum of the individual is about 7.3 pg*day / mL. In some embodiments, the dose of the CD-2 binding molecule is about 3 mg and administered intravenously, wherein the mean area under the curve (AUCiast) of the CD-2 binding molecule in the serum of the individual is about 6.8 pg*day / mL. In some embodiments, the dose of the CD-2 binding molecule is about 10 mg and administered by intramuscular injection, wherein the mean area under the curve (AUCiast) of the CD-2 binding molecule in the serum of the individual is about 23 pg*day / mL. In some embodiments, the dose of the CD-2 binding molecule is about 30 mg and administered by intramuscular injection, wherein the mean area under the curve (AUCiast) of the CD-2 binding molecule in the serum of the individual is about 54 pg*day / mL.

[0102] In some embodiments, AUC is an estimated AUC extrapolated from time zero to infinity (AUCinf). In some embodiments, the area under the curve (AUCinf) is a mean value (e.g., geometric mean). In some embodiments, the dose of the CD-2 binding molecule is about 1 mg and administered by intramuscular injection, wherein the mean area under the curve (AUCinf) of the CD-2 binding molecule in the serum of the individual is about 2.1 pg*day / mL. In some embodiments, the dose of the CD-2 binding molecule is about 3 mg and administered by intramuscular injection, wherein the mean area under the curve (AUCinf) of the CD-2 binding molecule in the serum of the individual is about 7.9 pg*day / mL. In some embodiments, the dose of the CD-2 binding molecule is about 3 mg and administered intravenously, wherein the mean area under the curve (AUCinf) of the CD-2 binding molecule in the serum of the individual is about 6.5 pg*day / mL. In some embodiments, the dose of the CD-2 binding molecule is about 10 mg and administered by intramuscular injection, wherein the mean area under the curve (AUCinf) of the CD-2 binding molecule in the serum of the individual is about 24 pg*day / mL. In some embodiments, the dose of the CD-2 binding molecule is about 30 mg and administered by intramuscular injection, wherein the mean area under the curve (AUCinf) of the CD-2 binding molecule in the serum of the individual is about 56 pg*day / mL.

[0103] In some embodiments, a dose of the CD-2 binding molecule provided herein is administered in about 0.5 ml to about 2.0 ml solution per injection. For instance, the CD-2 binding molecule is administered in about 0.2, about 1, about 1.5 or about 2.0 ml solution per injection.37MOFO-360045222Docket No.: 237752001540

[0104] In some embodiments, CD-2 binding molecule provided herein is administered in a single dose. In some embodiments, the CD-2 binding molecule is administered in a single dose of about 1 mg to about 30 mg. In some embodiments, the CD-2 binding molecule is administered in a single dose of about 3 mg to about 10 mg. In some embodiments, the CD-2 binding molecule is administered in a single dose of about 3 mg. In some embodiments, the CD-2 binding molecule is administered in a single dose of about 5 mg. In some embodiments, the CD-2 binding molecule is administered in a single dose of 7 mg. In some embodiments, the CD-2 binding molecule is administered in a single dose of about 10 mg.

[0105] In some embodiments, the individual is treated for a treatment period of up to 4 weeks, up to 5 weeks, up to 6 weeks, up to 7 weeks, up to 8 weeks, up to 9 weeks, up to 10 weeks, up to 11 weeks, up to 12 weeks, up to 13 weeks, up to 14 weeks, up to 15 weeks, up to 16 weeks, up to 17 weeks, up to 18 weeks, up to 19 weeks, up to 20 weeks, up to 21 weeks, up to 22 weeks, up to 23 weeks, up to 24 weeks, up to 25 weeks, up to 26 weeks, up to 27 weeks, up to 28 weeks, up to 29 weeks, up to 30 weeks, up to 31 weeks, up to 32 weeks, up to 33 weeks, up to 34 weeks, up to 35 weeks, up to 36 weeks, up to 37 weeks, up to 38 weeks, up to 39 weeks, up to 40 weeks, up to 41 weeks, up to 42 weeks, up to 43 weeks, up to 44 weeks, up to 45 weeks, up to 46 weeks, up to 47 weeks, up to 48 weeks, up to 49 weeks, up to 50 weeks, up to 51 weeks, or up to 52 weeks in length.C. CD2+cell Depletion

[0106] In some embodiments, methods of the present disclosure comprise administering a combination therapy comprising: 1) a CD-2 binding molecule comprising a CD2-binding domain of human leukocyte function-associated antigen 3 (LFA3) linked to an Fc domain provided herein and 2) a composition comprising modified regulatory T (Treg) cells, wherein the modified Treg cells express a therapeutic protein. In some embodiments, administration of the CD-2 binding molecule results in a reduction in the levels of CD2 positive cells in the individual. In some embodiments, the administration results in a reduction in the levels of CD2 positive cells in the individual as compared to the levels of CD2 positive cells in the individual prior to administration of the CD-2 binding molecule. In some embodiments, administration of the CD-2 binding molecule preconditions the individual for administration of the composition comprising modified Treg cells. In some embodiments, the composition comprising the modified Treg cells is administered after administration of the CD-2 binding molecule. In some embodiments, administration of the composition comprising the modified38MOFO-360045222Docket No.: 237752001540Treg cells is delayed to minimize depletion of the modified Treg cells by the CD-2 binding molecule.

[0107] The adhesion molecule CD2 (also known as LFA-2 for leukocyte function- associated antigen-2) is a transmembrane glycoprotein of the immunoglobulin superfamily expressed on the surface of T cells, NK cells, thymocytes and dendritic cells (DCs) (Binder C. et al., “CD2 Immunobiology” Fron. Immunol 2020, Krensky AM, “The functional significance, distribution, and structure ofLFA-1, LFA-2, and LFA-3: cell surface antigens associated with CTL-target interactions”. J Immunol. (1983), Matsui T, et al., “CD2 distinguishes two subsets of human plasmacytoid dendritic cells with distinct phenotype and functions. J Immunol. (2009)). CD2 functions as an intercellular adhesion molecule, binding to its ligand LFA-3 (also known as CD58) on antigen presenting cells (APCs). The interaction facilitates T cell receptor-binding and signal transduction.

[0108] In some embodiments, the CD2 positive cells are T cells. In some embodiments, the CD2 positive cells are Natural Killer (NK) cells. In some embodiments, the CD2 positive cells are thymocyte cells. In some embodiments, the CD2 positive cells are dendritic cells. In some embodiments, the CD2 positive cells are B cells. In some embodiments, the CD2 positive cells are effector T cells (Ten cells). In some embodiments, the CD2 positive (CD2+) cells are CD4 positive (CD4+) T cells. In some embodiments, the CD2 positive cells are CD8 positive T cells (CD8+). In some embodiments, the CD2 positive cells are CD8 positive Tcmcells. In some embodiments, the CD2 positive cells are effector memory T cells (Tem). In some embodiments, the CD2 positive cells are CD4+Temcells. In some embodiments, the CD2 positive cells are CD8+Temcells. In some embodiments, the CD2 positive cells are effector memory T cells re-expressing CD45RA. In some embodiments, the CD2 positive cells are CD8+Temra cells. In some embodiments, the CD2 positive cells are CD4+Temra cells. In some embodiments, the CD2 positive cells are naive T cells. In some embodiments, the CD2 positive cells are regulatory T cells (Treg).

[0109] In some embodiments, the CD2 positive cells express CD2 at various levels. In some embodiments, the effect of the CD-2 binding molecule provided herein on the CD2 positive cells depends on the levels of CD2 expression at the cell surface. For instance, CD2 is highly expressed on a subset of Teff cells (e.g., CD2hlghCD4+Temcells) as compared to Treg. In these aspects, the CD-2 binding molecule provided herein has differential depletive effects on the CD4+Temcells subtype, with selective sparing of Tregand naive T (e.g., naive CD4+Tnand naive CD8+Tn) cells.39MOFO-360045222Docket No.: 237752001540

[0110] In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of CD2 positive T cells in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of CD2 positive NK cells in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of CD2 positive thymocyte cells in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of CD2 positive dendritic cells in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of CD2 positive effector T cells (Teff cells) cells in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of CD2 positive CD4+positive T cells in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of CD2 positive CD8 positive T cells in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of CD2 positive central memory T cells (Tcmcells) in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of CD2 positive CD4 positive Tcmcells in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of CD2 positive CD8 positive Tcmcells in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of CD2 positive effector memory T cells (Tem) cells in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of CD2 positive cells are CD4+Temcells in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of CD2 positive cells are CD8+Temcells in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of CD2 positive cells are CD8+Tem cells in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of effector memory T cells reexpressing CD45RA in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of the CD2 positive cells are CD8+Temra cells in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of CD2 positive cells are CD4+Temra cells in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of effector memory T cells re-expressing40MOFO-360045222Docket No.: 237752001540CD45RA in the individual. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of naive T cells in the individual.[oni] In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of naive T cells in the individual. In some embodiments, the reduction in the levels of naive T cells (e.g., naive CD4+Tnand naive CD8+Tn) is not as high as the reduction in other T cell subtypes. In some embodiments, the levels of naive T cells (e.g., naive CD4+Tnand naive CD8+Tn) remain unchanged after administration of the CD-2 binding molecule as compared to the levels of the cells prior to administration. In some embodiments, administering a CD-2 binding molecule provided herein results in a reduction in the levels of regulatory T cells (Treg) in the individual. In some embodiments, the levels of regulatory T cells (Treg) remain unchanged after administration of the CD-2 binding molecule as compared to the levels of the cells prior to administration. In some embodiments, the reduction in the levels of regulatory T cells is not as high as the reduction in other T cell subtypes.

[0112] In some embodiments, administration of a CD-2 binding molecule comprising SEQ ID NO: 2 to the individual results in a reduction of CD4+Temcells of at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%, compared to the level of CD4+Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule. In some embodiments, the CD-2 binding molecule comprises SEQ ID NO: 2 and administration to the individual of one dose of about 1 mg of the CD-2 binding molecule results in a reduction of CD4+Tem in the peripheral blood of the individual by at least about 45% compared to the level of CD4+Temin the peripheral blood of the individual prior to administration of the CD- 2 binding molecule. In some embodiments, the CD-2 binding molecule comprises SEQ ID NO: 2 and administration to the individual of one dose of about 3 mg of the CD-2 binding molecule results in a reduction of CD4+Temin the peripheral blood of the individual by at least about 25% compared to the level of CD4+Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule. In some embodiments, the CD-2 binding molecule comprises SEQ ID NO: 2 and administration to the individual of one dose of about 3 mg of the CD-2 binding molecule results in a reduction of CD4+Temin the peripheral blood of the individual by at least about 45% compared to the level of CD4+Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule.41MOFO-360045222Docket No.: 237752001540In some embodiments, the CD-2 binding molecule comprises SEQ ID NO: 2 and administration to the individual of one dose of about 10 mg of the CD-2 binding molecule results in a reduction of CD4+Temin the peripheral blood of the individual by at least about 50% compared to the level of CD4+Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule. In some embodiments, the CD-2 binding molecule comprises SEQ ID NO: 2 and administration to the individual of one dose of about 30 mg of the CD-2 binding molecule results in a reduction of CD4+Temin the peripheral blood of the individual by at least about 80% compared to the level of CD4+Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule.

[0113] In some embodiments, administration of the CD-2 binding molecule provided herein to the individual results in a reduction of CD4+Temin the peripheral blood of the individual. In some embodiments, the reduction of CD4+Temis by a maximum change from baseline response value (Brmax) of at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% compared to the CD4+Tem cells in the peripheral blood of the individual prior to administration of the CD- 2 binding molecule provided herein. In some embodiments, administration of a CD-2 binding molecule comprising SEQ ID NO: 2 to the individual of one dose of about 1 mg of the CD-2 binding molecule provided herein results in a reduction of CD4+Temin the peripheral blood of the individual by a maximum change from baseline response value (Brmax) of at least about 80% compared to the level of CD4+Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule. In some embodiments, administration of a CD- 2 binding molecule comprising SEQ ID NO: 2 to the individual of one dose of about 3 mg of the CD-2 binding molecule provided herein results in a reduction of CD4+Temin the peripheral blood of the individual by a maximum change from baseline response value (Brmax) of at least about 75% compared to the level of CD4+Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule. In some embodiments, administration of a CD-2 binding molecule comprising SEQ ID NO: 2 to the individual of one dose of about 10 mg of the CD-2 binding molecule provided herein results in in a reduction of CD4+Temin the peripheral blood of the individual by a maximum change from baseline response value (Brmax) of at least about 80% compared to the level of CD4+Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule. In some embodiments, administration of a CD-2 binding molecule comprising SEQ ID NO: 242MOFO-360045222Docket No.: 237752001540 to the individual of one dose of about 30 mg of the CD-2 binding molecule provided herein results in a reduction of CD4+Temin the peripheral blood of the individual by a maximum change from baseline response value (Brmax) at least about 95% compared to the level of CD4+Tem in the peripheral blood of the individual prior to administration of the CD-2 binding molecule.

[0114] In some embodiments, administration of the CD-2 binding molecule provided herein to the individual results in an increase in the ratio of T regulatory cells (Treg) to CD4 positive Tem cells compared to the ratio of Tregto CD4 Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule provided herein. In some embodiments, the ratio of Tregto CD4 positive Temcells is increased by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, 150%, 200%, 250%, 260% 300%, 350%, 400%, 450%, or 500% compared to the ratio of Tregto CD4 Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule provided herein. In some embodiments, administration of a CD-2 binding molecule comprising SEQ ID NO: 2 to the individual of one dose of about 1 mg of the CD-2 binding molecule provided herein results in an increase in the ratio of Tregto CD4 Temin the peripheral blood of the individual by at least about 50% compared to the ratio of Tregto CD4 Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule provided herein. In some embodiments, administration of a CD-2 binding molecule comprising SEQ ID NO: 2 to the individual of one dose of about 3 mg of the CD-2 binding molecule provided herein results in an increase in the ratio of Tregto CD4 Temin the peripheral blood of the individual by at least about 10% compared to the ratio of Tregto CD4 Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule provided herein. In some embodiments, administration of a CD-2 binding molecule comprising SEQ ID NO: 2 of one dose of about 10 mg of CD-2 binding molecule provided herein results in an increase in the ratio of Tregto CD4 Tem in the peripheral blood of the individual by about at least about 100% compared to the ratio of Tregto CD4 Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule provided herein. In some embodiments, administration of a CD-2 binding molecule comprising SEQ ID NO: 2 to the individual of one dose of about 30 mg of the CD-2 binding molecule provided herein results in an increase in the ratio of Tregto CD4 Tem in the peripheral blood of the individual by at least about 260% compared to the ratio of43MOFO-360045222Docket No.: 237752001540Tregto CD4 Tem in the peripheral blood of the individual prior to administration of the CD-2 binding molecule provided herein.

[0115] In some embodiments, the AUECnet(3-36) is determined for cell populations. As described in, e.g., Example 1, AUECnet(3-36) can describe the net area under the effect curve from day 3 (2 days postdose) to day 36 (35 days postdose) using the percent change from baseline based on cell counts or cell population ratios. In some embodiments, Eavg, 3-36, i.e., the net average effect between day 3 and day 36, can be calculated as the AUECnet(3-36) divided by duration of time. In some embodiments, the AUECnet(3-8) is determined for cell populations. As described in, e.g., Example 1, AUECnet(3-8) can describe the net area under the effect curve from day 3 (2 days postdose) to day 8 (7 days postdose) using the percent change from baseline based on cell counts or cell population ratios. In some embodiments, Eavg, 3-8, i.e., the net average effect between day 3 and day 8, can be calculated as the AUECnet(3-36) divided by duration of time.

[0116] In some embodiments, administration of the CD-2 binding molecule provided herein to the individual results in an increase in the AUECnet(3-36) ratio of T regulatory cells (Treg) to CD4+Tem cells to compared to the AUECnet(3-36) ratio of Tregto CD4+Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule provided herein. In some embodiments, the AUECnet(3-36) ratio of Tregto CD4+Temcells is increased by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, 150%, 200%, 250%, 260% 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 740%, 800%, 850%, 900%, 1000%, 2000%, 3000%, 4000%, 5000%, 6000%, 7000%, 8000%, 9000%, 10000%, 11000%, 12000%, 13000%, 14000%, 15000%, 16000%, 17000%, 18000%, 19000%, 20000%, 21000%, 22000%, 23000%, 24000%, or 25000% compared to the AUECnet(3-36) ratio of Treg to CD4 Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule provided herein. In some embodiments, administration of a CD-2 binding molecule comprising SEQ ID NO: 2 to the individual of one dose of about 1 mg of the CD-2 binding molecule provided herein results in an increase in the AUECnet(3-36) ratio of Treg to CD4+Tem in the peripheral blood of the individual by at least about 4000 h*% compared to the AUECnet(3-36) ratio of Tregto CD4+Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule provided herein. In some embodiments, administration of a CD-2 binding molecule comprising SEQ ID NO: 2 to the44MOFO-360045222Docket No.: 237752001540 individual of one dose of about 3 mg of the CD-2 binding molecule provided herein results in an increase in the AUECnet(3-36) ratio of Tregto CD4+Temin the peripheral blood of the individual by at least about 10000 h*% compared to the AUECnet(3-36) ratio of Tregto CD4+Tem in the peripheral blood of the individual prior to administration of the CD-2 binding molecule provided herein. In some embodiments, administration of a CD-2 binding molecule comprising SEQ ID NO: 2 of one dose of about 10 mg of CD-2 binding molecule provided herein results in an increase in the AUECnet(3-36) ratio of Tregto CD4+Temin the peripheral blood of the individual by about at least about 7000 h*% compared to the AUECnet(3-36) ratio of Tregto CD4+Temin the peripheral blood of the individual prior to administration of the CD-2 binding molecule provided herein. In some embodiments, administration of a CD-2 binding molecule comprising SEQ ID NO: 2 to the individual of one dose of about 30 mg of the CD-2 binding molecule provided herein results in an increase in the AUECnet(3-36) ratio of Tregto CD4+Tem in the peripheral blood of the individual by at least about 20000 h*% compared to the AUECnet(3-36) ratio of CD4+Temto Tregin the peripheral blood of the individual prior to administration of the CD-2 binding molecule provided herein.

[0117] In some embodiments, the dose of the CD-2 binding molecule is about 1 mg and the area under the effect curve (AUECnet 3-36) of Treg / CD4+Tem in the peripheral blood of the individual is about 4000 h*%. In some embodiments, the dose of the CD-2 binding molecule is about 3 mg and the area under the effect curve (AUECnet 3-36) of Treg / CD4+Tem in the peripheral blood of the individual is about 10000 h*%. In some embodiments, the dose of the CD-2 binding molecule is about 10 mg and the area under the effect curve (AUECnet 3-36) of Treg / CD4+Temin the peripheral blood of the individual is about 7000 h*%. In some embodiments, the dose of the CD-2 binding molecule is about 30 mg and the area under the effect curve (AUECnet 3-36) of Treg / CD4+Tem in the peripheral blood of the individual is about 20000 h*%. Area under the effect curve (AUEC) is the integral of the effect of a drug over time. The AUEC reflects collective pharmacological contribution of the in vivo drug concentrations over a dosing interval.

[0118] In some embodiments, CD2 positive cells are circulating cells. In some embodiments, CD2 positive cells are present in the peripheral blood of the individual.

[0119] In some embodiments, the levels of the cells, including CD4+Temand / or Tregare measured in a sample of blood obtained from the individual. Any suitable assay may be used to detect, enumerate, determine phenotype, assess function and / or monitor the cells, and include, but are not limited to ELISPOT, ELISA, flow cytometry, assays assessing binding to45MOFO-360045222Docket No.: 237752001540 the antigen or ligand, or any other suitable assay. In some embodiments, the effects of the CD-2 binding molecule provided herein are measured by assessing the effect of the CD-2 binding on pharmacodynamic biomarkers, including but not limited to effector T cell depletion. In some embodiments, the effects of the CD-2 binding molecule provided herein are measured by evaluating the clinical outcome of the patient (e.g., changes in vital signs, pharmacokinetic assessments, pharmacodynamic assessments, clinical laboratory assessments, and changes in exploratory biomarkers).

[0120] In some embodiments, the method further comprises measuring the level of cells (e.g., CD4+Tem and / or Treg) in a sample of blood obtained from the individual before and / or after the individual has received or one or more doses of the CD-2 binding molecule provided herein. In some embodiments, the method further comprises measuring the level of cells in a sample of blood obtained from the individual before and after the individual has received a dose of the CD-2 binding molecule provided herein. In some embodiments, the level of cells in a sample of blood obtained from the individual is measured about 1 hour, 12 hours, 1 day, 2 days, 4 days, or 1 week prior to administration of the CD-2 binding molecule to the individual. In some embodiments, the level of cells in a sample of blood obtained from the individual is measured no more than 1 day, 2 day, 4 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, or 52 weeks prior to administration of the CD-2 binding molecule to the individual. In some embodiments, the level of cells in a sample of blood obtained from the individual is measured intermittently, e.g., at a frequency of about three times per week, twice per week, once per week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, or once every eight weeks after the dose of the CD-2 binding molecule is administered to the individual. In some embodiments, the level of cells in a sample of blood obtained from the individual is measured once after the dose of the CD-2 binding molecule is administered to the individual. In some embodiments, the level of cells in a sample of blood obtained from the individual is measured once after the dose of the CD-2 binding molecule is administered to the individual, wherein the level of cells in a sample of blood is measured at46MOFO-360045222Docket No.: 237752001540 least 1 hour, 12 hours, 1 day, 2 days, 4 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, or 20 weeks after the dose of the CD-2 binding molecule is administered.

[0121] In some embodiments, the method further comprises measuring the level of one or more inflammatory cytokines in a sample of blood obtained from the individual before and after the individual has received one or more doses of the CD-2 binding molecule provided herein. For instance, inflammatory cytokines, including but not limited to interferon gamma (IFN-y), interleukin-lbeta (IL-ip), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin- 10 (IL-10), interleukin-12p70 (IL-12p70), interleukin- 13 (IL-13), and tumor necrosis factor alpha (TNF-a) may be assessed by any suitable methods in the art.

[0122] In some embodiments, the method further comprising performing immunophenotyping of white blood cells in a sample of blood obtained from the individual before and after the individual has received one or more doses of the CD-2 binding molecule provided herein. Circulating cells in the blood can be immunophenotyped by any suitable method, including but not limited to, using antibodies directed against specific surface proteins and performing flow cytometry and / or microscopy, to detect the presence or absence of cell markers (e.g., cell surface markers). The process of immunophenotyping identifies the cells and / or cell subtypes by their known single or multiple markers (e.g., cell surface or intracellular protein markers, cytokine release, activation states).

[0123] In some embodiments, a CD-2 binding molecule comprising SEQ ID NO: 2 binds to CD2 and mediates at least one detectable activity selected from the following: (a) binding to CD2+cells, e.g., CD2-expressing memory CD4+T cells (CD4+Tmem cells) or CD2- expressing CD8+Tmem cells, (b) reduces the interaction between CD2 and a naturally occurring LFA3 ligand, (c) mediates cytotoxicity against CD2-expressing cells, e.g., CD2- expressing CD4+Tmem cells or CD2-expressing CD8+Tmem cells, e.g., in the presence of NK cells, (d) decreases CD4+and / or CD8+Temcells, (e) increases the Treg / Temratio, e.g., in CD4+and / or CD8+T cells, (f) increases the Treg / Tcmratio, e.g., in CD4+and / or CD8+T cells,(g) inhibits allogeneic T cell response, e.g., T cell proliferation and cytokine production, and(h) inhibits tetanus toxoid recall response. In some embodiments, memory T cells (Tmem) cells include, for example, central memory (Tcm) and effector memory (Tem) T cells.47MOFO-360045222Docket No.: 237752001540

[0124] In some embodiments, a CD-2 binding molecule comprising SEQ ID NO: 2 mediates cytotoxicity against CD2-expressing cells, e.g., CD4+Tmem cells, with an EC50 that is no more than about 400, 600, 800, 1000, 1200, 1400, or 1500 pM. In some embodiments, the CD-2 binding molecule provided herein mediates cytotoxicity against CD2- expressing cells, e.g., CD4+Tmem cells, with an EC50 of about 348 pM.

[0125] In some embodiments, a CD-2 binding molecule comprising SEQ ID NO: 2 mediates cytotoxicity against CD2-expressing cells, e.g., CD8+Tmem cells, with an EC50 that is no more than about 1, 5, 10, 20, 30, 40 or 50 nM. In some embodiments, CD-2 binding molecule provided herein mediates cytotoxicity against CD2-expressing cells, e.g., CD8+Tmem cells, with an EC50 of about 0.716 nM.

[0126] In some embodiments, a CD-2 binding molecule comprising SEQ ID NO: 2 mediates cytotoxicity against CD2-expressing cells, e.g., CD4+T non-memory cells (Tnon- mem), with an EC50 that is no more than about 1200, 1500, 1800, 2000, 2500, 3000, 3500, 4000 pM. In some embodiments, CD-2 binding molecule provided herein mediates cytotoxicity against CD2-expressing cells, e.g., CD4+TnOn-mem cells, with an EC50 of about 1256 pM.

[0127] In some embodiments, a CD-2 binding molecule comprising SEQ ID NO: 2 mediates cytotoxicity against CD2-expressing cells, e.g., CD8+Tnon-mem cells, with an EC50 that is no more than about 1, 5, 10, 20, 30, 40 or 50 nM. In some embodiments, CD-2 binding molecule provided herein mediates cytotoxicity against CD2-expressing cells, e.g., CD8+Tnon-mem cells, with an EC50 of about 0.787 nM.

[0128] In some embodiments, a CD-2 binding molecule comprising SEQ ID NO: 2 inhibits the allogenic response with an IC50 that is no more than about 400, 800, 1200, 1600, 2000, or 2400pM. In some embodiments, a CD-2 binding molecule comprising SEQ ID NO: 2 inhibits the allogenic response with an IC50 of about 302pM. In some embodiments, a CD-2 binding molecule comprising SEQ ID NO: 2 inhibits IFNy production of CD4+memory T cells in a tetanus toxoid recall response assay with an IC50 that is no more than about 5, 10, 15, 20, or 25nM. In some embodiments, a CD-2 binding molecule comprising SEQ ID NO: 2 inhibits IFNy production of CD4+memory T cells in a tetanus toxoid recall response assay with an IC50 of about 1.342 nM.48MOFO-360045222Docket No.: 237752001540IV. Methods of Treatment

[0129] The present disclosure provides methods of treating an autoimmune disease or inflammatory disease in an individual, comprising administering to the individual a) about 1 mg to about 30 mg of a CD-2 binding molecule comprising a CD2 -binding domain of human leukocyte function-associated antigen 3 (LFA3) linked to an Fc domain; and b) a composition comprising modified regulatory T (Treg) cells, wherein the modified Treg cells express a therapeutic protein; and wherein the composition comprising the modified Treg cells is administered after administration of the CD-2 binding molecule.

[0130] In some embodiments, the CD-2 binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 2, or a functional variant thereof. In some embodiments, the individual is a human subject.

[0131] In some embodiments, the autoimmune disease or inflammatory disease is mediated by CD2. Immune diseases, disorders, or conditions mediated by CD2 are those where modulation of CD2-expressing cells may have beneficial effects in the treatment of the autoimmune disease. As disclosed herein, the combination therapy provided may be used to treat an individual with an autoimmune disease or inflammatory disease mediated by CD2- expressing cells (e.g., CD2-expressing Temcells). In some embodiments, an individual has an improved response from a treatment comprising the CD-2 binding molecule and the composition comprising the modified Treg cells, as compared to the response of an individual who received the CD-2 binding molecule alone. In some embodiments, an individual has an improved response from a treatment comprising the CD-2 binding molecule and the composition comprising the modified Treg cells, as compared to the response of an individual who received the composition comprising the modified Treg cells alone.

[0132] In some embodiments, the autoimmune disease or inflammatory disease is characterized and / or mediated by immune cells that are positive for CD2. In such diseases, the CD-2 binding molecules provided herein can bind to the CD2 molecules expressed on the cell surface and alter the activation state, inhibit proliferation, and / or affect function of the CD2 positive cell, thus preconditioning the individual for administration of the composition comprising the modified Treg cells. For instance, the CD-2 binding molecules provided herein can bind to CD2 expressed on the surface of Teff cells and induce exhaustion or anergy of the overactive or autoreactive Teff cells. In some embodiments, the LFA-3 and Fc components of the CD-2 binding molecules provided herein can deplete CD2 positive cells49MOFO-360045222Docket No.: 237752001540 via antibody-dependent cellular cytotoxicity (ADCC). The IgGl domain of the CD-2 binding molecules provided herein can bind receptors (e.g., FCRylll) on innate immune cells such as NK cells to induce apoptosis of the CD2 positive cells via ADCC. The CD-2 binding molecules provided herein can act as a bridge between the CD2 positive cell and the innate immune cell. In some embodiments, the CD-2 binding molecules provided herein bind to CD2 molecules, modulate CD2-LFA3 interaction, and selectively deplete the CD2- expressing cells (e.g., memory T cells). In some aspects, diseases or disorders related to CD2 expression, include but are not limited to type- 1 -diabetes (T1D), plaque psoriasis, malignancies mediated by CD2 positive immune cells such as T cell malignancies or T cell lymphoma (e.g., cutaneous T- cell lymphoma or peripheral T-cell non-Hodgkin's lymphoma, NK cell malignancies, B cell malignancies, rheumatoid arthritis (RA), juvenile RA, systemic lupus erythematosus, ankylosing spondylitis, multiple sclerosis, or lyme disease. In some aspects, the condition is organ transplantation. In some aspects, the combination therapy may be used to prolong graft survival and minimize allograft rejection.

[0133] In some aspects, the present disclosure provides methods of treating an autoimmune disease or inflammatory disease in an individual, comprising administering to the individual: a) about 1 mg to about 30 mg of a CD-2 binding molecule comprising a CD2-binding domain of human leukocyte function-associated antigen 3 (LFA3) linked to an Fc domain; and b) a composition comprising modified regulatory T (Treg) cells, wherein the modified Treg cells express a therapeutic protein; and wherein the composition comprising the modified Treg cells is administered after administration of the CD-2 binding molecule. In some embodiments, the CD-2 binding molecule comprises SEQ ID NO: 2 and the disease, disorder, condition, or injury is an autoimmune disease or inflammatory disease. Autoimmune diseases or inflammatory disease include, but are not limited to, type 1 diabetes, psoriasis, plaque psoriasis, palmoplantaris pustulosis, pustular psoriasis of palms and soles, pustulosis palmaris et plantaris, pustulosis of palms and soles, atopic dermatitis, lichen planus, graft- versus-host disease (GVHD), vitiligo, Pityriasis Rubra Pilaris, transplantation (e.g., organ transplantation, e.g., kidney transplantation), psoriatic arthritis, a disease, disorder, or condition requiring allogeneic hematopoietic stem cell transplantation, thalassemia, sickle cell disease, glanzmann thrombasthenia, Wiskott-Aldrich syndrome, chronic-granulomatous disease, severe congenital neutropenia, leukocyte adhesion deficiency, Schwachman-Diamond syndrome, Diamond-Blackfan anemia, Fanconi anemia, Dyskeratosis-congenita, Chediak- Higashi syndrome, aplastic anemia, alopecia areata, and T cell lymphoma (e.g., cutaneous T-50MOFO-360045222Docket No.: 237752001540 cell lymphoma or peripheral T-cell non-Hodgkin's lymphoma). Additional exemplary diseases, disorders or conditions are: diabetes mellitus (e. g. Type I diabetes mellitus or insulin dependent diabetes mellitus); juvenile onset diabetes; inflammatory responses such as inflammatory skin diseases including psoriasis and dermatitis (e. g. atopic dermatitis); dermatomyositis; systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; gastritis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions involving infiltration of T cells and chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE); multiple sclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes typically found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; Wegener’s disease; pernicious anemia (Addison's disease); diseases involving leukocyte diapedesis; central nervous system (CNS) inflammatory disorder; multiple organ injury syndrome; hemolytic anemia (including, but not limited to cryoglobinemia or Coombs positive anemia); myasthenia gravis; antigen-antibody complex mediated diseases; anti-glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert- Eaton myasthenic syndrome; pemphigoid bullous; pemphigus; autoimmune polyendocrinopathies; vitiligo; Reiter's disease; stiff-person syndrome; Bechet disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathies; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia and autoimmune hemolytic diseases; Hashimoto’s thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune uveoretinitis; Guillain- Barre syndrome; Goodpasture's syndrome; mixed connective tissue disease; autoimmune- associated infertility; polyarteritis nodosa; alopecia areata; idiopathic myxedema; graft versus host disease; muscular dystrophy (Duchenne, Becker, Myotonic, Limb-girdle, Facioscapulohumeral, Congenital, Oculopharyngeal, Distal, Emery- Dreifuss); and an inflammatory non-immune disease, such as a heart disease or a brain disease. In some embodiments, the autoimmune disease or inflammatory disease is selected from the group consisting of type I diabetes, rheumatoid arthritis, hidradenitis suppurativa, amyotrophic lateral sclerosis, celiac disease, and inflammatory bowel disease.51MOFO-360045222Docket No.: 237752001540

[0134] A characteristic feature of patients with an autoimmune disease or inflammatory disease is the presence of effector T (Tetr) cells strongly responsive to antigenic stimulation and comparatively resistant to suppression by regulatory T cells (Treg) or downregulation by immune-modulating agents. For example, in patients with type I diabetes (T1D), persistence of autoreactive Teff contributes to the difficulty in preventing disease progression in new- onset T1D and maintaining allogeneic islet transplants by regular immunosuppressive regimens. Immune interventions that have demonstrated success in preserving P cell function in the new-onset period have been shown to deplete or modulate Teff (Rigby et al., Clin Invest. 2015; Diggins et al., JCI Insight 2021). Without wishing to be bound by theory, it is believed that, by targeting CD2+Teff, the CD-2 binding molecules provided herein have the therapeutic potential to prevent pancreatic P cell destruction and delay or even prevent the onset of T1D (Bluestone et al., Science. 2021). In some embodiments, the CD-2 binding molecules provided herein deplete Teff cells and spare the Tregand T naive cells to re-establish immune balance, slow beta-cell destruction with a goal of eliminating the need for exogenous insulin, decreasing the rate of major hypoglycemia, restoration of beta-cells, and ultimately reducing TID-related mortality.

[0135] In some embodiments, the CD-2 binding molecules provided herein are used to eliminate and / or suppress pathogenic T effector cells. In some embodiments, the CD-2 binding molecules provided herein are used to modulate the function of and selective depletion of CD2+ Temcells to rebalance the regulatory T cell (Treg) to Temcell numbers.

[0136] In some embodiments, the CD-2 binding molecules provided herein eliminate and / or suppress target cells via FcR-mediated antibody dependent cellular cytotoxicity (ADCC). In some embodiments, the CD-2 binding molecules provided herein eliminate and / or suppress target cells via apoptosis.

[0137] In some embodiments, the methods of treatment provided herein comprise administering to the individual a CD-2 binding molecule comprising SEQ ID NO: 2 at a dose of about 1 mg. In some embodiments, the methods of treatment provided herein comprise administering to the individual a CD-2 binding molecule comprising SEQ ID NO: 2 at a dose of about 3 mg. In some embodiments, the methods of treatment provided herein comprise administering to the individual a CD-2 binding molecule comprising SEQ ID NO: 2 at a dose of about 10 mg. In some embodiments, the methods of treatment provided herein comprise administering to the individual a CD-2 binding molecule comprising SEQ ID NO: 2 at a dose of about 30 mg.52MOFO-360045222Docket No.: 237752001540

[0138] In some embodiments, the methods of treatment provided herein comprise administering a CD-2 binding molecule, comprising SEQ ID NO: 2 to an individual, at a dose of about 1 mg to about 30 mg per administration (e.g., injection). For instance, the CD-2 binding molecule is administered at a dose of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, or about 30 mg per administration (e.g., injection).

[0139] In some embodiments, the methods of treatment provided herein comprise administering a CD-2 binding molecule comprising SEQ ID NO: 2 to an individual by an intramuscular injection.

[0140] In some embodiments, the methods of treatment provided herein comprise administering a CD-2 binding molecule comprising SEQ ID NO: 2 to an individual intravenously.

[0141] In some aspects, provided herein is a method of treating an autoimmune disease or inflammatory disease in an individual comprising administering to the individual: a) about 1 mg to about 30 mg of a CD-2 binding molecule comprising a CD2 -binding domain linked to an Fc domain; and b) a composition comprising modified regulatory T (Treg) cells, wherein the modified Treg cells express a therapeutic protein; and wherein the composition comprising the modified Treg cells is administered after administration of the CD-2 binding molecule. In some embodiments, the method further comprises isolating T cells from a biological sample obtained from the individual prior to administration of the CD-2 binding molecule. In some embodiments, the method further comprises enriching the T cells for Treg cells; transfecting the enriched Treg cells with an expression vector encoding the therapeutic protein; and expanding the transfected Treg cells; wherein expansion produces the modified Treg cells of the composition; and wherein enrichment, transfection, and expansion are performed prior to administration of the composition comprising the modified Treg cells. In some embodiments, the expansion comprises using anti-CD3 / CD28 coated beads. In some embodiments, the expansion does not comprise using anti-CD3 / CD28 coasted beads. In some embodiments, the transfection occurs by use of a viral vector, electroporation, heat shock, bacteriophage, sonication, or calcium phosphate.53MOFO-360045222Docket No.: 237752001540V. Pharmaceutical Compositions

[0142] Provided herein are pharmaceutical compositions and / or pharmaceutical formulations for use in the treatment of an autoimmune disease or inflammatory disease.

[0143] In some embodiments, a pharmaceutical composition and / or formulation comprises the CD-2 binding molecule (e.g., LF-vl or functional variant thereof) of the present disclosure and a pharmaceutically acceptable carrier or excipient. The disclosure also provides pharmaceutical compositions comprising an effective amount of a CD-2 binding molecule provided herein. Examples of such compositions, as well as how to formulate, are also described herein. In some embodiments, the composition comprises one or more such CD-2 binding molecules.

[0144] The disclosure also provides compositions, including pharmaceutical compositions, comprising polynucleotides encoding the CD-2 binding molecule of the disclosure. In some embodiments, the composition comprises an expression vector comprising a polynucleotide encoding the CD-2 binding molecule provided herein.

[0145] In some embodiments, a pharmaceutical composition and / or formulation comprises the modified Treg cells expressing a therapeutic protein of the present disclosure and a pharmaceutically acceptable carrier or excipient. The disclosure also provides pharmaceutical compositions comprising an effective amount of the modified Treg cells. Examples of such compositions, as well as how to formulate, are also described herein. In some embodiments, the composition comprises one or more than one type of modified Treg cell, for example the composition comprises two types of modified Treg cells each expressing a different therapeutic protein. In some embodiments, the composition comprises an expression vector comprising a polynucleotide encoding the therapeutic protein of the modified Treg cells.

[0146] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutical acceptable excipient" includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, HEPES (4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid) buffered saline, water, emulsions such as oil / water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's54MOFO-360045222Docket No.: 237752001540Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000). To prepare pharmaceutical or sterile compositions including the CD-2 binding molecule, the CD-2 binding molecule is mixed with a pharmaceutically acceptable carrier or excipient. To prepare pharmaceutical or sterile compositions including the modified Treg cells, the modified Treg cells are mixed with a pharmaceutically acceptable carrier or excipient. Formulations of therapeutic and diagnostic agents can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N. Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

[0147] The pharmaceutical composition used in the present disclosure can further comprise pharmaceutically acceptable carriers, excipients, or stabilizers (Remington: The Science and practice of Pharmacy 20th Ed., 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations, and may comprise buffers such as phosphate, citrate, HEPES and other organic acids. In some embodiments, pharmaceutical compositions comprising CD-2 binding molecules provided herein, for example LF-vl polypeptide comprising SEQ ID NO: 2, include HEPES-buffered saline. In some embodiments, a concentration of a HEPES buffer is about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM. In some embodiments, a concentration of HEPES buffer is 20 mM. Additional acceptable carriers, excipients or stabilizers include antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins;55MOFO-360045222Docket No.: 237752001540 hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter- ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0148] The pharmaceutical compounds of the disclosure may include one or more pharmaceutically acceptable salts. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

[0149] A pharmaceutical composition of the disclosure also may include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil- soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BEIT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, acetic acid, phosphoric acid, and the like.

[0150] Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical compositions of the disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.56MOFO-360045222Docket No.: 237752001540

[0151] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0152] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be suitable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0153] In some embodiments, the CD-2 binding molecules of this present disclosure are formulated for subcutaneous injection. In some embodiments, the CD-2 binding molecules of this present disclosure are formulated for intramuscular injection. In some embodiments, the CD-2 binding molecules of this present disclosure are formulated for intravenous administration. In some embodiments, the CD-2 binding molecules of this present disclosure are formulated for intravascular administration.

[0154] In some embodiments, the compositions comprising modified Treg cells of the present disclosure are formulated for intravenous administration. In some embodiments, the compositions comprising modified Treg cells of the present disclosure are formulated for intravascular administration.57MOFO-360045222Docket No.: 237752001540

[0155] Cells can be cryopreserved. Cryopreservation can include formulating cells with a cryopreservation agent, such as DMSO. Commercially available media include, for example, CryoStor® and pZerve®, available from Millipore Sigma.

[0156] In certain embodiments, the CD-2 binding molecules of the present disclosure are formulated to ensure proper distribution in vivo.VI. Nucleic Acids, Vectors, and Host CellsA. CD-2 Binding Molecules

[0157] Provided herein are polynucleotides encoding any of the CD-2 binding molecules described herein. The invention also provides a method of making any of the CD-2 binding molecule provided herein. The CD-2 binding molecules provided herein can be made and expressed by procedures known in the art.

[0158] The sequence of a desired CD-2 binding molecule, and nucleic acid encoding such CD-2 binding molecule, or portion thereof, can be determined using standard sequencing techniques. A nucleic acid sequence encoding a desired CD-2 binding molecule may be inserted into various vectors (such as cloning and expression vectors) for recombinant production and characterization.

[0159] Provided herein is a polynucleotide comprising the nucleic acid sequence as set forth in SEQ ID NO: 1. In some embodiments, a polynucleotide encoding an amino acid sequence, encodes an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and more preferably identical to, the amino acid sequence of a CD-2 binding molecule provided herein. In some embodiments, a polynucleotide encoding an amino acid sequence encodes an amino acid sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and more preferably identical to, the amino acid sequence of SEQ ID NO:2.

[0160] Further provided herein are host cells comprising one or more nucleic acid molecules comprising SEQ ID NO: 1.

[0161] In another aspect, the present disclosure provides polynucleotides and variants thereof encoding a CD-2 binding molecule provided herein, wherein such variant polynucleotides share at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of the specific nucleic acid sequences disclosed herein. In some embodiments, the invention provides58MOFO-360045222Docket No.: 237752001540 polynucleotides and variants thereof, encoding CD-2 binding molecules provided herein, wherein such variant polynucleotides share at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. These amounts are not meant to be limiting, and increments between the recited percentages are specifically envisioned as part of the disclosure.

[0162] Polynucleotides complementary to any such sequences are also encompassed by the present disclosure. Polynucleotides may be single-stranded (coding or antisense) or doublestranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present disclosure, and a polynucleotide may, but need not, be linked to other molecules and / or support materials.

[0163] Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes the CD-2 binding molecule provided herein) or may comprise a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and / or insertions such that the immunoreactivity of the encoded CD-2 binding molecule provided herein is not diminished, relative to a native immunoreactive molecule. The effect on the immunoreactivity of the encoded CD-2 binding molecule provided herein may generally be assessed as described herein. In some embodiments, variants exhibit at least about 70% identity, in some embodiments, at least about 80% identity, in some embodiments, at least about 90% identity, and in some embodiments, at least about 95% identity to a polynucleotide sequence that encodes the CD-2 binding molecule provided herein. These amounts are not meant to be limiting and increments between the recited percentages are specifically envisioned as part of the disclosure.

[0164] It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a CD-2 binding molecule provided herein as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present disclosure. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present disclosure. Alleles are endogenous genes59MOFO-360045222Docket No.: 237752001540 that are altered as a result of one or more mutations, such as deletions, additions and / or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and / or database sequence comparison).

[0165] The polynucleotides of this disclosure can be obtained using chemical synthesis, recombinant methods, or PCR. Methods of chemical polynucleotide synthesis are well- known in the art and need not be described in detail herein. One of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to produce a desired DNA sequence. For preparing polynucleotides using recombinant methods, a polynucleotide comprising a desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification, as further discussed herein. Polynucleotides may be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome. The polynucleotide so amplified can be isolated from the host cell by methods well-known within the art. See, e.g., Sambrook et al, 1989. Alternatively, PCR allows reproduction of DNA sequences. PCR technology is well- known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al eds., Birkauswer Press, Boston, 1994. RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well-known to those of skill in the art, as set forth in Sambrook et al, 1989, for example.

[0166] In some embodiments, a vector comprises a polynucleotide that encodes the CD-2 binding molecule provided herein. In some embodiments, the vector is transfected into host cells.

[0167] In some embodiments, a vector is selected that is optimized for expression of a CD- 2 binding molecule provided herein in CHO or CHO-derived cells, or in NSO cells.Exemplary such vectors are described, e.g., in Running Deer et al, Biotechnol. Prog. 20:880- 889 (2004).60MOFO-360045222Docket No.: 237752001540

[0168] Suitable cloning and expression vectors can include a variety of components, such as promoter, enhancer, and other transcriptional regulatory sequences. The vector may also be constructed to allow for subsequent cloning of an antibody variable domain into different vectors. Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and / or may carry genes for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen. Expression vectors are further provided. Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide according to the disclosure. It is implied that an expression vector must be replicable in the host cells either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector(s) disclosed in PCT Publication No. WO 87 / 04462. Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional controlling elements (such as promoters, enhancers and terminator). For expression (i.e., translation), one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.

[0169] The vectors containing the polynucleotide encoding the CD-2 binding molecule provided herein, can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of introducing vectors or polynucleotides will often depend on features of the host cell.

[0170] The CD-2 binding molecule provided herein can be made recombinantly using a suitable host cell. A nucleic acid encoding the CD-2 binding molecule provided herein can be61MOFO-360045222Docket No.: 237752001540 cloned into an expression vector, which can then be introduced into a host cell, such as E. coli cell, a yeast cell, an insect cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell where the cell does not otherwise produce an immunoglobulin protein, to obtain the synthesis of the CD-2 binding molecule provided herein in the recombinant host cell. Preferred host cells include a CHO cell, a Human embryonic kidney HEK-293 cell, or an Sp2.0 cell, among many cells well-known in the art. Methods of chemical synthesis for proteins and peptides are known in the art and are commercially available.

[0171] In various embodiments, the CD-2 binding molecule provided herein is expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used to express the CD-2 binding molecule provided herein include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E and Expi293 cells; CHO cells, including CHO-S, DG44. Lecl3 CHO cells, ExpiCHO and FUT8 CHO cells; PER.C6® cells (Crucell); and NSO cells. In some embodiments, the CD-2 binding molecule provided herein is expressed in yeast. See, e.g., U.S. Publication No. US 2006 / 0270045 Al. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the CD-2 binding molecule provided herein. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.

[0172] Introduction of one or more nucleic acids into a desired host cell may be accomplished by any method, including but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Nonlimiting exemplary methods are described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001). Nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.

[0173] The CD-2 binding molecule provided herein may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography, e.g., a Protein A, Protein G, or Protein A / G. Hydrophobic interactive chromatography, for example, a butyl or phenyl column, may also suitable for purifying the CD-2 binding molecule provided herein. Many methods of purifying polypeptides are known in the art.62MOFO-360045222Docket No.: 237752001540

[0174] In some embodiments, the CD-2 binding molecule provided herein is produced in a cell-free system. Non-limiting exemplary cell- free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al, Biotechnol. Adv. 21 : 695-713 (2003).VII. Kits / Articles of Manufacture

[0175] Provided herein are articles of manufacture (e.g., kit) comprising the CD-2 binding molecule provided herein and / or the composition comprising the modified Treg cells. Article of manufacture may include one or more containers comprising the CD-2 binding molecule provided herein. Article of manufacture may include one or more containers comprising the composition comprising the modified Treg cells. In some embodiments, the article of manufacture comprises the CD-2 binding molecule provided herein and the composition comprising the modified Treg cells in separate containers. Containers may be any suitable packaging including, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.

[0176] In some embodiments, the kit contains instructions for administering to the individual a) about 1 mg to about 30 mg of a CD-2 binding molecule comprising a CD2- binding domain of human leukocyte function-associated antigen 3 (LFA3) linked to an Fc domain; and b) a composition comprising modified regulatory T (Treg) cells, wherein the modified Treg cells express a therapeutic protein; and wherein the composition comprising the modified Treg cells is administered after administration of the CD-2 binding molecule. In some embodiments, the kit contains instructions for administering to the individual a composition comprising modified regulatory T (Treg) cells, wherein the modified Treg cells express a therapeutic protein, and wherein the therapeutic protein is a chimeric antigen receptor (CAR). In some embodiments, the kit contains instructions for administering to the individual a CD-2 binding molecule comprising SEQ ID NO: 2 at a dose of about 1 mg to about 30 mg. In some embodiments, the kit contains instructions for administering to the individual a CD-2 binding molecule comprising SEQ ID NO: 2 at a dose of about 1 mg. In some embodiments, the kit contains instructions for administering to the individual a CD-2 binding molecule comprising SEQ ID NO: 2 at a dose of about 3 mg. In some embodiments, the kit contains instructions for administering to the individual a CD-2 binding molecule comprising SEQ ID NO: 2 at a dose of about 10 mg. In some embodiments, the kit contains63MOFO-360045222Docket No.: 237752001540 instructions for administering to the individual a CD-2 binding molecule comprising SEQ ID NO: 2 at a dose of about 30 mg.VIII. Terminology

[0177] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0178] It is understood that aspect and embodiments of the invention described herein include "consisting" and / or “consisting essentially of aspects and embodiments. As used herein, the singular form "a", "an", and "the" includes plural references unless indicated otherwise.

[0179] In this application, the use of "or" means "and / or" unless expressly stated or understood by one skilled in the art. In the context of a multiple dependent claim, the use of "or" refers back to more than one preceding independent or dependent claim. Throughout this specification and claims, the word "comprise," or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Any example(s) following the term “e.g.,” or “for example” is not meant to be exhaustive or limiting.

[0180] It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of and / or “consisting essentially of are also provided.

[0181] Polypeptide "fragments" or “portions” according to the invention may be made by truncation, e.g., by removal of one or more amino acids from the N and / or C-terminal ends of a polypeptide. Up to 10, up to 20, up to 30, up to 40 or more amino acids may be removed from the N and / or C terminal in this way. Fragments may also be generated by one or more internal deletions.64MOFO-360045222Docket No.: 237752001540

[0182] A variant molecule may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions and / or insertions from the specific sequences and fragments discussed above. "Deletion" variants may comprise the deletion of individual amino acids, deletion of small groups of amino acids such as 2, 3, 4 or 5 amino acids, or deletion of larger amino acid regions, such as the deletion of specific amino acid domains or other features. "Insertion" variants may comprise the insertion of individual amino acids, insertion of small groups of amino acids such as 2, 3, 4 or 5 amino acids, or insertion of larger amino acid regions, such as the insertion of specific amino acid domains or other features. "Substitution" variants preferably involve the replacement of one or more amino acids with the same number of amino acids. Substitutions may be conservative or nonconservative. Variant molecule may comprises any combination of deletions, insertions, and substitutions.

[0183] Non-conservative substitutions are made by exchanging a member of one of these classes for another class.

[0184] One type of substitution, for example, that may be made is to change one or more cysteines in the molecule, which may be chemically reactive, to another residue, such as, without limitation, alanine or serine. For example, there can be a substitution of a non- canonical cysteine. In some embodiments, the cysteine is canonical. Any cysteine residue not involved in maintaining the proper conformation of the molecule also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bond(s) may be added to the molecule to improve its stability.

[0185] Lymphocyte-function-associated antigen 3” or “LFA3,” used interchangeably herein, also referred to in the art as “CD58” is a member of the immunoglobulin superfamily. The term LFA3 includes LFA3 homologs and orthologs, including human, cynomolgus monkey, rat, rabbit, and mouse, among others. As used herein, “LFA3” refers to a mammalian LFA3, such as human, rat or mouse, as well as non-human primate, bovine, ovine, or porcine LFA3. A non- limiting example of LFA3 is human LFA3 (see, e.g., UniProtKB Accession Number P19256, SEQ ID NO: 2). The term “LFA3” also encompasses fragments, variants, isoforms, and other homologs of such LFA3 molecules. Variant LFA3 molecules will generally be characterized by having the same type of activity as naturally occurring LFA3, such as the ability to bind CD2.65MOFO-360045222Docket No.: 237752001540

[0186] A “LFA3 domain” refers to a fragment of LFA3, or a variant thereof, which is a ligand of CD2. In some embodiments, the LFA3 domain is no more than 100, 110, 120, 130, 140, 150, 160, 170, or 180 amino acids in length. In some embodiments, the LFA3 domain is derived from the first extracellular domain of LFA3. In some embodiments, the LFA3 domain comprises no more than 6, 10, 15, 20, or 30 amino acid mutations (e.g., substitutions, additions, or deletions) relative to the wild type LFA3 sequence.

[0187] “ CD2,” also referred to in the art as “erythrocyte receptor, “LFA-3 receptor,” “rosette receptor,” or “T-cell surface antigen T1 l / Leu-5” is a molecule expressed on cells such as T cells and NK cells. The term CD2 includes CD2 homologs and orthologs, including human, cynomolgus monkey, rat, rabbit, and mouse, among others. As used herein, “CD2” refers to a mammalian CD2, such as human, rat or mouse, as well as non-human primate, bovine, ovine, or porcine CD2. A non- limiting example of CD2 is human CD2 (see, e.g., UniProtKB Accession Number P06729). The term “CD2” also encompasses fragments, variants, isoforms, and other homologs of such CD2 molecules. Variant CD2 molecules will generally be characterized by having the same type of activity as naturally occurring CD2.

[0188] All ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10.

[0189] The term "identity," as known in the art, refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or nucleic acid molecule sequences, as the case may be, as determined by the match between strings of nucleotide or amino acid sequences. "Identity" measures the percent of identical matches between two or more sequences with gap alignments addressed by a particular mathematical model of computer programs (i. e. "algorithms"). A molecule that “preferentially binds” or “specifically binds” (used interchangeably herein) to a target is a term well understood in the art, and methods to determine such specific or preferential binding are also well-known in the art. A molecule is said to exhibit “specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular cell or substance than it does with alternative cells or substances. A molecule “specifically66MOFO-360045222Docket No.: 237752001540 binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. Also, a molecule “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration to that target in a sample than it binds to other substances present in the sample. For example, a molecule that specifically or preferentially binds to CD2 is a molecule that binds CD2 with greater affinity, avidity, more readily, and / or with greater duration than it binds to a non-CD2 protein. It is also understood by reading this definition, for example, that a molecule which specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding. “Specific binding” or “preferential binding” includes a compound, e.g., a protein, a nucleic acid, and the like, which recognizes and binds to a specific molecule, but does not substantially recognize or bind other molecules in a sample. For instance, a molecule which recognizes and binds to a cognate ligand or binding partner (e.g., a polypeptide molecule that binds CD2) in a sample but does not substantially recognize or bind other molecules in the sample, specifically binds to that cognate ligand or binding partner. Thus, under designated assay conditions, the specified binding moiety binds preferentially to a particular target molecule and does not bind in a significant amount to other components present in a test sample.

[0190] A " fusion" protein or “fusion” molecule is a protein wherein a first polypeptide is operably linked, e.g., directly or indirectly, to a second polypeptide. In some embodiments, a CD-2 binding molecule provided herein is a fusion protein comprising a LFA3 domain operably linked to a second polypeptide.

[0191] An "Fc fusion" protein or “Fc fusion” molecule is a protein wherein one or more polypeptides are operably linked, e.g., directly or indirectly, to an Fc polypeptide. An Fc fusion comprises the Fc region of an immunoglobulin with a fusion partner. In some embodiments, the CD-2 binding molecule provided herein is an Fc fusion protein comprising a LFA3 domain operably linked to an Fc polypeptide.

[0192] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g. NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with67MOFO-360045222Docket No.: 237752001540 cytotoxins. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII, and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Pat. Nos. 5,500,362 or 5,821,337 or U.S. Pat. No. 6,737,056 (Presta), may be performed. Useful effector cells for such assays include PBMC and NK cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998). Additional antibodies with altered Fc region amino acid sequences and increased or decreased ADCC activity are described, e.g., in U.S. Pat. No. 7,923,538, and U.S. Pat. No. 7,994,290.

[0193] As known in the art, “polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to chains of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the chain. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0194] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutical acceptable excipient" includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil / water emulsion, and various types of wetting agents. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).

[0195] As used herein, "vector" means a construct, which is capable of delivering, and, preferably, expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with68MOFO-360045222Docket No.: 237752001540 cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0196] A “host cell” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected and / or transformed in vivo with a polynucleotide of this invention. Host cells may be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Any host cell susceptible to cell culture, and to expression of protein or polypeptides, may be utilized in accordance with the present invention. In certain embodiments, the host cell is mammalian. Mammalian cell lines available as hosts for expression are well-known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). Nonlimiting exemplary mammalian cells include, but are not limited to, NSO cells, HEK 293 and Chinese hamster ovary (CHO) cells, and their derivatives, such as 293 -6E and CHO DG44 cells, CHO DXB11, and Potelligent® CHOK1SV cells (BioWa / Lonza, Allendale, NJ). Mammalian host cells also include, but are not limited to, human cervical carcinoma cells (HeLa, ATCC CCL 2), baby hamster kidney (BHK, ATCC CCL 10) cells, monkey kidney cells (COS), and human hepatocellular carcinoma cells (e.g., Hep G2). Other non-limiting examples of mammalian cells that may be used in accordance with the present invention include human retinoblasts (PER.C6®; CruCell, Leiden, The Netherlands); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line 293 (HEK 293) or 293 cells subcloned for growth in suspension culture (Graham et a , 1977, J. Gen Virol. 36:59); mouse sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et ak, 1982, Annals N.Y. Acad. Sci. 383:44-68); MRC 5 cells; LS4 cells; a human hepatoma line (Hep G2); and numerous myeloma cell lines, including, but not limited to, B ALB / c mouse myeloma line (NS0 / 1, ECACC No: 85110503), NSO cells and Sp2 / 0 cells. Additionally, any number of commercially and non-commercially available cell lines that69MOFO-360045222Docket No.: 237752001540 express polypeptides or proteins may be utilized in accordance with the present invention. One skilled in the art will appreciate that different cell lines might have different nutrition requirements and / or might require different culture conditions for optimal growth and polypeptide or protein expression, and will be able to modify conditions as needed.

[0197] As used herein, “treatment” is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: improved survival rate (reduced mortality), reduction in inflammatory response to the disease, reduction in the amount of tissue fibrosis, improvement in the appearance of the disease lesions, limitation of the pathological lesions to focal sites, decreased extent of damage from the disease, decreased duration of the disease, and / or reduction in the number, extent, or duration of symptoms related to the disease. The term includes the administration of the combination therapy the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.

[0198] “Ameliorating” means a lessening or improvement of one or more symptoms as compared to not administering a combination therapy comprising a LFA3 polypeptide molecule and a composition comprising modified regulatory T (Treg) cells. “Ameliorating” also includes shortening or reduction in duration of a symptom.

[0199] As used herein, an “effective dosage” or “effective amount” of drug, compound, or pharmaceutical composition is an amount sufficient to affect any one or more beneficial or desired results. In more specific aspects, an effective amount prevents, alleviates or ameliorates symptoms of disease or infection, and / or prolongs the survival of the subject being treated. For prophylactic use, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histological and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms of a disease, disorder or condition, decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication, and / or delaying the70MOFO-360045222Docket No.: 237752001540 progression of the disease of patients. An effective dosage can be administered in one or more administrations. For purposes of this invention, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective dosage” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

[0200] An “individual” or a "subject" is a mammal, more preferably, a human. In certain embodiments, the subject has an autoimmune or inflammatory disease, disorder or condition. In certain embodiments, the subject is in need of immunosuppression therapy.

[0201] As used herein, “autoimmune disease” refers to a disease characterized by immune activity that targets an individual’s healthy cells, tissues, and / or organs. An autoimmune disease may be characterized by overactive immune cells, for example by overactive macrophages, eosinophils, mast cells, effector T cells, etc. In some instances, regulatory immune cells (e.g., regulatory T cells (Treg)) that provide inhibitory signals to immune cells (e.g., effector T cells) may be underactive or otherwise unable to effectively inhibit effector immune cells. As a result, autoimmune diseases are often accompanied by inflammation (e.g., inflammation localized within the affected organ system and / or systemic inflammation). In some instances, an individual suffering from one autoimmune disease may have an increased risk of developing one or more additional autoimmune diseases, for example type 1 diabetes and celiac disease wherein the two common HLA haplotypes (i.e., DR4-DQ8 and DR3-DQ2) lead to increased risk of developing both of these autoimmune diseases.

[0202] As used herein, “inflammatory disease” refers to a disease in which the level of inflammation is pathologically high and triggers disease progression, often by recruiting immune cells to the inflamed tissue or organ. Non-limiting examples of inflammatory diseases include allergy, asthma, autoimmune diseases, coeliac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, reperfusion injury, and transplant rejection.

[0203] As used herein, the terms “antigen,” “immunogen,” and “antibody target,” refer to a molecule, compound, or complex that is recognized by an antibody, i.e., can be bound by the antibody. The term can refer to any molecule that can be recognized by an antibody, e.g., a71MOFO-360045222Docket No.: 237752001540 polypeptide, polynucleotide, carbohydrate, lipid, chemical moiety, or combinations thereof (e.g., phosphorylated or glycosylated polypeptides, etc.). One of skill will understand that the term does not indicate that the molecule is immunogenic in every context, but simply indicates that it can be targeted by an antibody.

[0204] As used herein, the term “epitope” refers to the localized site on an antigen that is recognized and bound by an antibody. Epitopes can include a few amino acids or portions of a few amino acids, e.g., 5 or 6, or more, e.g., 20 or more amino acids, or portions of those amino acids. In some cases, the epitope includes non-protein components, e.g., from a carbohydrate, nucleic acid, or lipid. In some cases, the epitope is a three-dimensional moiety. Thus, for example, where the target is a protein, the epitope can be comprised of consecutive amino acids, or amino acids from different parts of the protein that are brought into proximity by protein folding (e.g., a discontinuous epitope).

[0205] As used herein, the term “antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene, that specifically bind and recognize an antigen. Typically, the “variable region” contains the antigen-binding region of the antibody (or its functional equivalent) and is most critical in specificity and affinity of binding. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD).

[0206] Antibodies can be of (i) any of the five major classes of immunoglobulins, based on the identity of their heavy-chain constant domains - alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) and mu (IgM), or (ii) subclasses (isotypes) thereof (E.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2). The light chains can be either lambda or kappa.

[0207] The following are a non-exhaustive list of different antibody forms, all retaining antigen binding activity: (1) whole immunoglobulins (also referred to as “intact” antibodies) (two light chains and two heavy chains, e.g., a tetramer); (2) an immunoglobulin polypeptide (a light chain or a heavy chain); (3) an antibody fragment, such as Fv (a monovalent or bivalent variable region fragment, and can encompass only the variable regions e.g., VL and / or VH), Fab (VLCL VHCH), F(ab')2, Fv (VLVH), SCFV (single chain Fv) (a polypeptide comprising a VL and VH joined by a linker, e.g., a peptide linker), (scFv)2, sc(Fv)2, bispecific sc(Fv)2, bispecific (scFv)2, minibody (sc(FV)2 fused to CH3 domain), diabody (noncovalent dimer of single-chain Fv (scFv) fragment that consists of the heavy chain variable (VH) and light72MOFO-360045222Docket No.: 237752001540 chain variable (VL) regions connected by a small peptide linker), triabody is trivalent sc(Fv)3 or trispecific sc(Fv)3; (4) a multivalent antibody (an antibody comprising binding regions that bind two different epitopes or proteins, e.g., “scorpion” antibody; (5) a fusion protein comprising a binding portion of an immunoglobulin fused to another amino acid sequence (such as a fluorescent protein); and (6) heavy chain only antibody or antibody fragment having only two heavy chains and lacking the two light chains usually found in antibodies.

[0208] Production and properties of tandem scFvs and diabodies are described, e.g., in Asano et al. (2011) J Biol. Chem. 286: 1812; Kenanova et al. (2010) Prot Eng Design Sei 23:789; Asano et al. (2008) Prot Eng Design Sei 21 :597.EXEMPLARY EMBODIMENTS

[0209] Exemplary embodiments of the methods described herein include:1. A method of treating an autoimmune disease or inflammatory disease in an individual comprising administering to the individual a) about 1 mg to about 30 mg of a CD-2 binding molecule comprising a CD2-binding domain of human leukocyte function-associated antigen 3 (LFA3) linked to an Fc domain; and b) a composition comprising modified regulatory T (Treg) cells, wherein the modified Treg cells express a therapeutic protein; wherein the composition comprising the modified Treg cells is administered after administration of the CD-2 binding molecule.2. The method of embodiment 1, wherein about 3 mg to about 10 mg of the CD-2 binding molecule is administered to the individual.3. The method of embodiment 1 or 2, wherein about 3 mg of the CD-2 binding molecule is administered to the individual.4. The method of embodiment 1 or 2, wherein about 5 mg of the CD-2 binding molecule is administered to the individual.5. The method of embodiment 1 or 2, wherein about 7 mg of the CD-2 binding molecule is administered to the individual.73MOFO-360045222Docket No.: 2377520015406. The method of embodiment 1 or 2, wherein about 10 mg of the CD-2 binding molecule is administered to the individual.7. The method of any one of embodiments 1-6, wherein the CD-2 binding molecule is administered by intramuscular injection.8. The method of any one of embodiments 1-7, wherein about 3 mg, 5 mg, 7 mg, or 10 mg of the CD-2 binding molecule is administered to the individual, and the CD-2 binding molecule is administered by intramuscular injection.9. The method of any one of embodiments 1-6, wherein the CD-2 binding molecule is administered intravenously.10. The method of any one of embodiments 1-3 and 9, wherein about 3 mg of the CD-2 binding molecule is administered to the individual and the CD-2 binding molecule is administered intravenously.11. The method of any one of embodiments 1-10, wherein the LFA3 domain comprises an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 3.12. The method of any one of embodiments 1-11, wherein the LFA3 domain comprises the amino acid sequence of SEQ ID NO: 3.13. The method of any one of embodiments 1-12, wherein the Fc domain comprises an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 4.14. The method of any one of embodiments 1-13, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO: 4.74MOFO-360045222Docket No.: 23775200154015. The method of any one of embodiments 1-14, wherein the CD-2 binding molecule comprises an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 2.16. The method of any one of embodiments 1-15, wherein the CD-2 binding molecule comprises the amino acid sequence of SEQ ID NO: 2.17. The method of any one of embodiments 1-16, wherein the composition comprising the modified Treg cells is administered about 50 days to about 120 days after administration of the CD-2 binding molecule.18. The method of any one of embodiments 1-17, wherein the composition comprising the modified Treg cells is administered about 80 days to about 90 days after administration of the CD-2 binding molecule.19. The method of any one of embodiments 1-18, wherein administration of the CD-2 binding molecule to the individual results in a reduction of CD4+ effector memory T-cells (Tern) in the peripheral blood of the individual.20. The method of embodiment 19, wherein the composition comprising the modified Treg cells is administered when the level of CD4+ Tern in the peripheral blood of the individual is reduced by at least about 40% compared to the level of CD4+ Tern in the peripheral blood of the individual prior to administration of the CD-2 binding molecule.21. The method of embodiment 19, wherein the composition comprising the modified Treg cells is administered when the level of CD4+ Tern in the peripheral blood of the individual is reduced by about 25% to about 50% compared to the level of CD4+ Tern in the peripheral blood of the individual prior to administration of the CD-2 binding molecule.22. The method of any one of embodiments 1-21, wherein administration of the CD-2 binding molecule to the individual results in a reduction of CD4+ central memory T-cells (Tcm) in the peripheral blood of the individual.75MOFO-360045222Docket No.: 23775200154023. The method of embodiment 22, wherein the composition comprising the modified Treg cells is administered when the level of CD4+ Tcm in the peripheral blood of the individual is reduced by at least about 40% compared to the level of CD4+ Tcm in the peripheral blood of the individual prior to administration of the CD-2 binding molecule.24. The method of embodiment 22, wherein the composition comprising the modified Treg cells is administered when the level of CD4+ Tcm in the peripheral blood of the individual is reduced by about 30% to about 50% compared to the level of CD4+ Tcm in the peripheral blood of the individual prior to administration of the CD-2 binding molecule.25. The method of any one of embodiments 1-24, wherein the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the serum of the individual is less than about 50 ng / mL.26. The method of any one of embodiments 1-25, wherein the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the serum of the individual is less than about 20 ng / mL.27. The method of any one of embodiments 1-24, wherein the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the serum of the individual is about 10 ng / mL to about 50 ng / mL.28. The method of any one of embodiments 1-27, wherein the composition comprising the modified Treg cells is administered intravenously.29. The method of any one of embodiments 1-3, 7, 8, 11-17, and 19-28, wherein about 3 mg of the CD-2 binding molecule is administered to the individual, wherein the CD-2 binding molecule is administered by intramuscular injection, and wherein the composition comprising the modified Treg cells is administered about 65 days to about 80 days after administration of the CD-2 binding molecule.30. The method of any one of embodiments 1-2, 4, 7, 8, 11-17, and 19-28, wherein about 5 mg of the CD-2 binding molecule is administered to the individual, wherein the CD-2 binding molecule is administered by intramuscular injection, and wherein the composition76MOFO-360045222Docket No.: 237752001540 comprising the modified Treg cells is administered about 75 days to about 95 days after administration of the CD-2 binding molecule.31. The method of any one of embodiments 1-2, 5, 7, 8, 11-17, and 19-28, wherein about 7 mg of the CD-2 binding molecule is administered to the individual, wherein the CD-2 binding molecule is administered by intramuscular injection, and wherein the composition comprising the modified Treg cells is administered about 80 days to about 105 days after administration of the CD-2 binding molecule.32. The method of any one of embodiments 1-2, 6, 7, 8, 11-17, and 19-28, wherein about 10 mg of the CD-2 binding molecule is administered to the individual, wherein the CD-2 binding molecule is administered by intramuscular injection, and wherein the composition comprising the modified Treg cells is administered about 85 days to about 120 days after administration of the CD-2 binding molecule.33. The method of any one of embodiments 1-3, 9-17, and 19-28, wherein about 3 mg of the CD-2 binding molecule is administered to the individual, wherein the CD-2 binding molecule is administered intravenously, and wherein the composition comprising the modified Treg cells is administered about 55 days to about 80 days after administration of the CD-2 binding molecule.34. The method of any one of embodiments 1-33, wherein the therapeutic protein is a T cell receptor (TCR) comprising an antigen-binding site that specifically binds to antigen.35. The method of any one of embodiments 1-33, wherein the therapeutic protein is a chimeric antigen receptor (CAR).36. The method of any one of embodiments 1-35, wherein the modified Treg cells are CD4+, CD25+ and CD1271o.37. The method of embodiment 35, wherein the CAR comprises an antigen binding domain that specifically binds to an antigen that is associated with an autoimmune disease or inflammatory disease.77MOFO-360045222Docket No.: 23775200154038. The method of embodiment 37, wherein the antigen is selected from the group consisting of MHC class I, MHC class II, collagen type V, K alphal tubulin, MHC class I related chain A (MICA), fibronectin, collagen type IV, collagen type VI, vimentin, angiotensin II type 1 receptor (AGTR1), perlecan, agrin, collagen type I, collagen type II, collagen type III, cardiac myosin, an islet cell autoantibody (ICA) antigen, insulin, glutamic acid decarboxylase (GAD), myelin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, astrocyte proteins, glial fibrillary protein (GFAP), SlOObeta, beta cell antigen, insulin B chain, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD65), islet-associated antigen 2, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), zinc transporter 8 (ZnT8), islet antigen 2 (IA-2), heat shock protein 60 (HSP60), chromogranin A, dnaJ (heat shock protein), citrullinated- vimentin, human cartilage glycoprotein-39, mitochondrial component, pyruvate dehydrogenase (mitochondrial), E2 component of pyruvate dehydrogenase, E2 component of branched chain 2-oxo acid dehydrogenase, E2 component of 2-oxo-glutarate dehydrogenase complex, E3 binding protein of dihydrolipoamide dehydrogenase, a nuclear component, nuclear protein splOO, nuclear pore complex protein gpl20, centromere, acetylcholine receptor (AChR), aquaporin-4 (AQP-4), CTLA-4, ICAM, LFA-3, CD40 / CD154, ICOS / ICOSL, CD52, nuclear factor of activated T cells (NF AT), phospholipase C (PLC), CD25, Janus kinase, B cell activating factor (BAFF), a proliferating inducing ligand (APRIL), IL6R, IL 17, IL12 / IL23, an integrin, a sphingosin receptor, melanocyte antigen, tolllike receptor, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, MyD88, an IL-lR-associated kinase (IRAK), an allergen associated with the allergic disorder, a citrullinated protein or citrullinated fragments thereof, citrullinated vimentin, and citrullinated filaggrin.39. The method of embodiment 37 or 38, wherein the antigen binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof.40. The method of any one of embodiments 37-39, wherein the antigen binding domain of the CAR specifically binds to citrullinated vimentin.41. The method of any one of embodiments 37-40, wherein the antigen binding domain of the CAR binds to all three of (i) citrullinated vimentin, (ii) citrullinated filaggrin, and (iii) citrullinated fibrinogen, or citrullinated peptides fragments thereof, optionally wherein the antigen binding domains further binds to citrullinated tenascin C.78MOFO-360045222Docket No.: 23775200154042. The method of any one of embodiments 1-41, wherein the modified Treg cells are human T cells.43. The method of any one of embodiments 1-42, wherein the modified Treg cells are primary T cells.44. The method of any one of embodiments 1-43, wherein the individual is a human.45. The method of any one of embodiments 1-44, wherein the autoimmune disease or inflammatory disease is a disease mediated by CD2.46. The method of any one of embodiments 1-45, wherein the autoimmune disease or inflammatory disease is selected from the group consisting of type I diabetes, rheumatoid arthritis, hidradenitis suppurativa, amyotrophic lateral sclerosis, celiac disease, and inflammatory bowel disease.

[0210] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and not intended to be limiting.EXAMPLES

[0211] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the invention, and not by way of limitation.Example 1: Randomized, Placebo-Controlled Phase I Study to Evaluate the Pharmacokinetics and Pharmacodynamics of Single Ascending Doses of LF-vl in Healthy Participants.

[0212] This Example provides results of a Phase 1 study in healthy participants to assess the pharmacokinetics (PK) and pharmacodynamics (PD) of administering LF-vl, an exemplary CD-2 binding molecule comprising the amino acid sequence of SEQ ID NO: 2. When treating a patient with a combination therapy comprising a CD-2 binding molecule and a Treg cell therapy of the disclosure, the PK and PD of the CD-2 binding molecule are important79MOFO-360045222Docket No.: 237752001540 parameters in determining the timing of Treg cell therapy infusion. The objective of the PD analysis was to assess the PD activity, using endogenous biomarkers as analytes after LF-vl single dose administration in healthy participants, and to evaluate the relationship with PK (PK / PD).Methods

[0213] As shown in FIG. 1 and FIG. 2, healthy participants were administered single ascending doses (SAD) across a broad range of doses of either LF-vl or matching placebo via intramuscular injection (IM) or intravenous (IV) 30-minute infusion. Approximately 40 participants were enrolled and randomized in up to 5 dose cohorts, with 8 participants included in each (6 participants received the study drug, while the other 2 received placebo). Cohort 1 participants were administered (IM) 1 mg of the study drug or placebo IM, Cohort 2 participants were administered (IM) 3 mg of the study drug or placebo IM, Cohort 3 participants were administered (IM) 10 mg of the study drug or placebo IM, Cohort 4 participants were administered (IM) 30 mg of the study drug or placebo IM, and Cohort 5 participants were administered (IV) 3 mg of the study drug or placebo IV. The study consisted of three parts: screening (up to 28 days), treatment period (1 day), and follow-up period (10 weeks for Cohorts 1-4 and 20 weeks for Cohort 5).

[0214] All participants had samples collected for PD assessments. Serial blood samples were collected from Cohorts 1 to 4 at the screening visit, on Day 1 at 0 hour (predose), at 2, 6, 8, 16 hours postdose, as well as on Day 2 (24 hours postdose), Day 3, Day 4, Day 8, Day 15, Day 22, and Day 36. Serial blood samples were collected in Cohort 5 at the screening visit, and on Day 1 at 0 hour (predose) and 2 hours postdose, as well as on Day 2, Day 3, Day 4, Day 8, Day 15, Day 22, Day 36, Day 43, Day 57, Day 78, and Day 99. Additional blood samples were taken at the end of study visit on Day 72 (Cohorts 1 to 4) or Day 143 (Cohort 5) or at early termination (no such case occurred), as well as any unscheduled visit.

[0215] The PD Analysis Set consisted of all participants who received a dose of active study drug or placebo and had at least one blood sample postdose collected to assess PD effects.

[0216] The PK / PD Analysis Set consisted of all participants who had at least one blood sample collected to assess PK or PD concentrations, had sufficient concentrations to estimate PK parameters (including participants who received placebo and had their PK parameter values set to 0) and sufficient values to estimate at least 1 PD parameter.80MOFO-360045222Docket No.: 237752001540

[0217] The Immunogenicity / PD Analysis Set consisted of all participants who received a dose of active study drug, had a baseline and at least one postdose immunogenicity assessment, and who had sufficient PD measurements to estimate at least 1 PD parameter.

[0218] All PBMC PD parameter calculations were performed using nominal times in days relative to start of administration (i.e., start of injection / infusion for IM / IV treatment). PD parameters were determined using noncompartmental analysis (NCA) methods based on individual observation-time data for the PD analytes listed in Table 1 below.Table 1: List of PD Analytes* B cell population includes CD3-CD19+ B cells Natural killer (NK) cell population includes CD3-CD56+ NK cells&T cell sub-populations include:1) CD3+T cells referred to as CD3+T cells,2) CD4+T cells referred to as CD4+T cells,3) TregCD4+FoxP3+CD25hlreferred to as Tregcells,4) CD4+ Tnawe CD4+FOXP3-CD45RA+CCR7+referred to as CD4+TnalVe cells,5) CD4+Tcm(central memoiy) CD4+FOXP3-CD45RA-CCR7+referred to as CD4+Tcmcells,6) CD4+Tem(effector memoiy) CD4+FOXP3-CD45RA-CCR7- referred to as CD4+Temcells,7) CD8+T cells referred to as CD8+T cells,8) CD8+T naive CD8+CD45RA+CCR7+referred to as CD8+Tnaive cells,9) CD8+TcmCD8+CD45RA-CCR7+referred to as CD8+Tcmcells,10) CD8+TemCD8+CD45RA-CCR7- referred to as CD8+Temcells, and11) CD8+Temra CD8+CD45RA+CCR7- referred to as CD8+Temracells.

[0219] PD parameters listed in Table 2 were determined using NCA methods based on individual observation-time profile. The areas under the effect-time curve (AUECs) were calculated using the linear trapezoidal method with linear interpolation.Table 2: List of Planned Pharmacodynamic Parameters81MOFO-360045222Docket No.: 237752001540

[0220] In addition, the cell population ratios listed in Table 3 were reported. For participants with observed absolute cell count values of 0 in CD4+Temcells, CD8+Temcells, CD4+Tcmcells, or CD8+Tcmcells, the absolute count for that cell populations at that time point was set to 1 for the calculation of ratios.Table 3: List of Cell Population Ratios

[0221] Individual absolute cell counts, % change from baseline of absolute cell counts for analytes and ratio of analytes (Table 3), and PD parameters were listed by the appropriate grouping factors (analyte, cohort, dose, time point), and were summarized with the following descriptive statistics: sample size (n), arithmetic mean (Mean), standard deviation (SD), coefficient of variation (CV%), median, range, QI, Q3, geometric mean and geometric CV%.

[0222] CD2 levels were reported to 2 decimals, except in groups where all individual values were reported as integer in the source data, in which cases 0 decimal places were used.Individual cell counts, % change from baseline of absolute cell counts for analytes, and cell population ratios were reported were reported to 2 decimals. Individual PD parameters were reported with at least 3 significant figures.

[0223] Reporting rules for the summary statistics were as follows:82MOFO-360045222Docket No.: 237752001540• N: integer• Mean and geometric mean: 1 more decimal / significant digit than the individual value• SD: 1 more decimal / significant digit than the mean• CV% and Geometric CV%: always 1 decimal• Median, Range, Min, Max, QI, and Q3: same number of decimals / significant digits as the individual value

[0224] Individual PD cell counts, % change from baseline for analytes and ratio of analytes were plotted vs. nominal sampling time on linear scales and ordinal scales. Mean PD observations (+SD) were plotted vs. nominal sampling time on both linear and ordinal scales using the appropriate grouping factors (analyte and dose). When grouping by dose, placebo participants from all five cohorts were pooled.Results

[0225] PK samples were collected for 40 participants, with 8 participants in the placebo IM group and 2 participants in the placebo IV group, as well as 6 participants in each active treatment group. All 40 participants were included in the PD Analysis Set and the PK / PD Analysis Set, and 32 participants (excluding participants who received placebo) were included in the Immunogenicity / PD Analysis Set.

[0226] FIGS. 3A-3B show mean (±SD) serum concentration of LF-vl versus time following a single intramuscular (IM) or intravenous (IV) administration of LF-vl on linear scale (FIG. 3A) and semi-log scale (FIG. 3B). Summaries of LF-vl PK parameters following single IM or IV administration of LF-vl stratified by treatment are presented in Table 4 below. Following a single IM administration of 1 to 30 mg LF-vl, maximum serum LF-vl levels were observed approximately 3 days post dose over the 1 to 10 mg dose range and 4 days post dose at 30 mg dose level. PK exposures increased when dose increased from 1 to 30 mg LF-vl in the IM cohorts. Following a single IV administration of 3 mg LF-vl, maximum serum LF-vl concentrations were detected at 0.083 day post dose (Hour 2), the first sampling timepoint after the end of infusion (EOI). In all participants, serum LF-vl levels declined in a biphasic manner with a faster initial distribution phase and a slower terminal elimination phase.83MOFO-360045222Docket No.: 237752001540Table 4: Geometric Mean Summary LF-vl PK ParametersMedian (Min; Max); NGeometric CV% = percent coefficient of variation of the geometric mean; IM = intramuscular; IV = intravenous;N = number of subjects with available information; NA = not applicable; PK = pharmacokinetic

[0227] The average observed drug concentrations in participants at Days 35, 42, 56, 71, and 77 are provided in Table 5 below. At Day 56, the observed drug concentration for the 3mg IV dose was less than 20 ng / mL indicating a marginal benefit in terms of pharmacokinetics and possible timing of Treg cell administration

[0228] Next, the levels of CD4+ T cm and CD4+ Temcells were measured in peripheral blood sampled from the study participants. FIGS. 4A-4D show the trends and variations in the T cell subset data in response to different doses of LF-vl in Cohorts 1-5. The percentage change of lymphocyte subsets from baseline was measured over the course of treatment through 1704 treatment hours (Cohorts 1-4) or 3,360 treatment hours (Cohort 5 and placebo). Overall, LF-vl caused a depletion of CD4+ Temand CD4+ Tcmcells in the peripheral blood of the participants.

[0229] FIGS. 5A-5J depict the decreases in absolute cell counts observed following 1 mg to 30 mg IM administration of the study drug. FIGS. 5A-5B show the mean (+SD) percent change from baseline of absolute count of CD4+Tcmcells on a numeric (FIG. 5A) and ordinal (FIG. 5B) scale. FIGS. 5C-5D show the mean (+SD) percent change from baseline84MOFO-360045222Docket No.: 237752001540 of absolute count of CD4+Temcells on a numeric (FIG. 5C) and ordinal (FIG. 5D) scale. FIGS. 5E-5F show the mean (+SD) percent change from baseline of absolute count of Tregcells on a numeric (FIG. 5E) and ordinal (FIG. 5F) scale. FIGS. 5G-5H show the mean (+SD) percent change from baseline of absolute count of CD4+Tnaive cells on a numeric (FIG. 5G) and ordinal (FIG. 5H) scale. FIGS. 5I-5J show the mean (+SD) percent change from baseline of absolute count of CD8+Temra cells on a numeric (FIG. 51) and ordinal (FIG. 5J) scale. FIGS. 5K-5L show the mean (+SD) percent change from baseline of absolute count of NK cells on a numeric (FIG. 5K) and ordinal (FIG. 5L) scale.

[0230] In general, PCFB of absolute cell counts were close to 0 following placebo administration, and greater decreases in absolute cell counts were observed at the higher doses in the LF-vl-treated groups. In those T cell sub-populations, maximum mean suppression of up to -75% from baseline was observed around Day 2 of dosing at the 1 mg and 3 mg dose levels and within about 8 hours of dosing at 10 mg and 30 mg dose levels.

[0231] At the 1 and 3 mg IM doses, absolute cell counts of CD3+T cells, CD4+T cells, CD4+Tcmcells, CD4+Temcells (Img IM only), CD8+T cells, CD8+Tcmcells (Img IM only), and CD8+Temcells (1 mg IM only) recovered from nadir, with some plateauing as early as Day 4 and other by Day 15, however those absolute cell counts remained below baseline throughout the observation period. In the 3 mg IM treated group, CD4+Temcell population and CD8+Temcells recovered and their PCFBs were above 0 by Day 15 while CD8+Tcmcell population remained suppressed by Day 72.

[0232] At the 10 mg dose, the absolute cell counts of CD3+T cells, CD4+T cells, and CD8+T cells recovered from nadir; the plateau phases began at different timepoints across cell populations, ranging from Day 14 to Day 15, and were sustained through Day 72 without reaching the baseline. The absolute cell counts of CD4+Tcmcells, CD4+Temcells, CD8+Tcmcells, and CD8+Temcells remained suppressed at around -50% by Day 72.

[0233] At the 30 mg IM dose level, absolute cell counts of CD3+T cells, CD4+T cells, CD4+Tcmcells, CD4+Temcells, CD8+T cells, CD8+Tcmcells, and CD8+Temcells remained suppressed throughout the study, with PCFB staying below -50% until the end of the collection period on Day 72.

[0234] Following 3 mg IV administration of the study drug, decrease in absolute cell counts was also observed in CD3+T cells, CD4+T cells, CD4+Tcmcells, CD4+Temcells, CD8+T cells, CD8+Tcm cells, and CD8+Temcells. Maximum mean decrease in PCFB was reached85MOFO-360045222Docket No.: 237752001540 more quickly than IM, occurring at 2 hours postdose, while the magnitude of the maximum suppression was similar to that observed in the 30 mg IM group. Although maximum mean suppression was similar, unlike the 30 mg IM group, PCFB following IV administration began increasing after Day 3 and was within 10% of baseline by Day 72.

[0235] Mean TregPCFB cell counts decreased after treatment with the study drug (FIGS. 5E-5F). Maximum suppression was observed at 2 hours postdose for the 3 mg IV group, and between Day 2 and Day 3 for the IM groups. Mean postdose PCFB in the placebo group ranged between -31.5% and 11.7%, while mean postdose PCFB in the LF-vl between -74.1% and 26.2%. Due to the large between-subject variability in response, no clear trends could be established between dose and response in the 1 to 10 mg IM groups. At the 30 mg IM dose level, Tregcells were suppressed throughout the study, with PCFB remaining around -50% by Day 72.

[0236] In CD4+Tnaive (FIGS. 5F-5G) and CD8+Tnaive cell populations, minimal to no change from baseline was observed at the 1 mg IM dose level. In higher dose groups (3 to 30 mg IM and 3 mg IV), a decrease from baseline in absolute cells counts was observed, with maximum suppression occurring within 3 to 4 days of dosing. Tnaive cell populations remained suppressed until the end of the study in the 30 mg IM dose groups, however in the 10 mg IM group CD4+Tnaive cell counts returned to near baseline levels by Day 15.

[0237] In the CD8+Temracells (FIGS. 5I-5J) and NK cells (FIGS. 5K-5L), following 1 mg to 30 mg of IM administration of LF-vl, maximum decrease in mean cell counts was observed between 8 to 24 hours postdose. For these two cell populations, the PCFB of absolute cell counts was significantly reduced by the end of the observation period post IM treatment. Following 3 mg IV administration of LF-vl, the decrease in mean cell counts was transient, and the maximum decrease was observed within 2 hours postdose.

[0238] In B cell population, minimal to no decrease in PCFB of cells counts was observed at the 1 mg and 3 mg IM dose level. In higher dose groups (10 to 30 mg IM and 3 mg IV), a decrease from baseline in absolute cells counts was observed, with maximum suppression occurring around 8 hours and 2 hours postdose for IM and IV groups, respectively. Mean PCFB of cell counts returned to near baseline levels (>-25%) by Day 8 in all groups.

[0239] To quantify how absolute levels of CD4+Temcells, CD8+Temcells, CD4+Tcmcells, and CD8+Tcmcells changed relative to Tregcells in response to LF-vl or placebo administration, the following cell ratios were calculated: Treg: CD4+Temratio , Treg: CD8+86MOFO-360045222Docket No.: 237752001540Tem ratio, Treg: CD4+Tcmratio, Treg: CD8+Tcmratio (the formula used is presented in Table 3). The PCFB of cell ratios were calculated by 100*(Cell ratio at time t - cell ratio at baseline) / cell ratio at baseline). FIGS. 6A-6B depict linear scales of the mean (+SD) percent change from baseline (PCFB) of CD4+cell ratios (Treg: CD4+Tcmratio (FIG. 6A) and Treg: CD4+Tem ratio (FIG. 6B)) over time by type and treatment (placebo or study drug). PCFB in all cell ratios were highly variable, with CV% values generally above 100%.

[0240] In the 1 mg IM group and placebo group, the mean PCFB of ratios between Tregcells and Tcm cells with phenotypic marker CD4+and CD8+were generally below the 0 suggesting that Tregvs Tcm ratio decreased compared to baseline (i.e., Tregcells have become less abundant relative to Tcmcells over time), whereas for the 3 mg to 30 mg IM and 3 mg IV groups, the mean PCFB ratios were generally above the 0 suggesting that Tregvs Tcmratio increased compared to baseline (i.e., Tregcells have become more abundant relative to Tcmcells over time). By the end of the observation period, mean PCFB of Treg: CD4+Tcmratios and Treg: CD8+Tcmratios ranged between -5.6% and 125.7% and between 84.5% and 222.7% for the 3 mg to 30 mg IM and 3 mg IV LF-vl-treated groups, respectively.

[0241] In the placebo group, the mean PCFB of ratios between Tregcells and Temcells with phenotypic marker CD4+and CD8+were similar to baseline. In the LF-vl groups, the mean PCFB of ratios between Tregcells and Temcells with phenotypic marker CD4+and CD8+were generally above the 0 suggesting that Tregvs Temratio increased compared to baseline (i.e., Tregcells have become more abundant relative to Temcells over time). By the end of the observation period, mean PCFB of Treg: CD4+Temratios and Treg: CD8+Temratios ranged between -15.4% and 444.0% and between -1.0% and 154.1% for the 3 mg to 30 mg IM and 3 mg IV LF-vl-treated groups, respectively.

[0242] Over the period of 72 days, ratios oscillate above and below 0 for the placebo group, suggesting dynamic shifts between Tregcells, Tcm, and Temcells. Dose-related trend increases in Treg: Tcmratios and Treg: Temratios were observed for the 1 mg, 10 mg and 30 mg IM groups.

[0243] Summary PD parameters of different cell populations and different cell population ratios are presented in Table 6 and Table 7, respectively. Following 1 mg to 30 mg of LF-vl IM single dose administration, dose-dependent average decreases (measured by Eavg, 3-8 and Eavg, 3-36) were observed across different cell populations: CD3+T cells, CD4+T cells, CD4+Tcmcells, CD4+Temcells, CD8+T cells, CD8+Tcmcells, CD8+Temcells, and NK cells. For87MOFO-360045222Docket No.: 237752001540Tregcells, B cells, Tnaive cells, and CD8+Temra cells, dose-related trends were harder to discern due to high individual variabilities, but suppression was still observed (Table 6). In general, the 3 mg IM group had a greater decrease, demonstrated by a lower or equivalent Eavg, 3-8 and Eavg, 3-36 values compared to the 3 mg IV group. The IM group showed greater and more sustained decreases in cell counts from Day 3 to Day 36 compared to the IV group, where counts returned to near baseline by Day 8 in most cell populations. Following 1 mg, 10 mg and 30 mg of LF-vl IM single dose administration, dose-dependent increase in Eavg, 3-8 and EaVg, 3-36 were observed across different cell population ratios: Treg :CD4+Temratio, Treg: CD8+Tem ratio, Treg: CD4+Tcmratio, and Treg: CD8+Tcmratio. An increase in Eavg, 3-8 and EaVg, 3-36 was al so observed in the 3 mg LF-vl IV group. The Eavg, 3-8 and Eavg, 3-36 of cell population ratios in the 3mg IM group had high individual variability, with relative standard deviation (RSD%) greater than 150 (Table 7). The high variability was mainly driven by a specific subject (Participant 014, who had unusually low Tregabsolute cells counts starting at Hour 0 compared to other participants in the same treatment, which led to high Eavg, 3-8 and EaVg, 3-36 of cell population ratios).Table 6: Arithmetic Mean and RSD Summary of PD Parameters of Different Cell Populations Following Study Drug Treatment or Placebo88MOFO-360045222Docket No.: 23775200154089MOFO-360045222Docket No.: 237752001540B = baseline; Eavg, 3-8 = average effect between Day 3 to Day 8; Eavg, 3-36 = average effect between Day 3 to Day 36; MFI = mean fluorescence intensity; RSD = relative standard deviation, calculated as the absolute value of (arithmetic standard deviation / arithmetic mean)Table 7: Arithmetic Mean and RSD Summary of PD Parameters of Different Cell Population Ratios Following Study Drug Treatment or Placebo90MOFO-360045222Docket No.: 237752001540B = baseline; Eavg, 3-8 = average effect between Day 3 to Day 8; Eavg, 3-36 = average effect between Day 3 to Day 36; RSD = relative standard deviation, calculated as the absolute value of (arithmetic standard deviation / arithmetic mean)

[0244] FIGS. 7A-7M and FIGS. 8A-8D depict Eavg, 3-36 of different cell populations following placebo or study drug treatment: Eavg, 3-36 of CD3+ T cells (FIG. 7A); Eavg, 3-36 of CD4+ T cells (FIG. 7B); Eavg,3-36 of Treg cells (FIG. 7C); Eavg,3-36 of CD4+ Tnaive cells (FIG. 7D); Eavg, 3-36 of CD4+ Tcm cells (FIG. 7E); Eavg, 3-36 of CD4+ Tern cells (FIG. 7F); Eavg, 3-36 of CD8+ T cells (FIG. 7G); Eavg, 3-36 of CD8+ Tnaive cells (FIG. 7H); Eavg, 3-36 of CD8+ Tcm cells (FIG. 71); Eavg,3-36 of CD8+ Tem cells (FIG. 7 J); Eavg, 3-36 of CD8+ Temra cells (FIG. 7K); Eavg, 3-36 ofNK cells (FIG. 7L); Eavg, 3-36 of B cells (FIG. 7M); Eavg, 3-36 of Treg : CD4+ Tcm ratio (FIG. 8A); Eavg, 3-36 of Treg : CD4+ Tem ratio (FIG. 8B); Eavg, 3-36 of Treg : CD8+ Tcm ratio (FIG. 8C); Eavg, 3-36 of Treg : CD8+ Tem ratio (FIG. 8D).

[0245] As shown in Table 6, the mean baseline (B) of absolute cell counts was similar across treatment groups for CD3+T cells, CD4+T cells, Tregcells, CD8+T cells, CD8+Tcmcells, NK cells, B cells. There were some variabilities in B between groups in CD4+Tnaive cells, CD4+Tcmcells, CD4+Temcells, CD8+Tnaive cells, CD8+Temcells, and CD8+Temracells.91MOFO-360045222Docket No.: 237752001540The mean B of cell population ratios was variable for all ratios: Treg :CD4+Temratio, Treg: CD8+Tem ratio, Treg: CD4+Tcmratio, and Treg: CD8+Tcmratio.

[0246] Boxplots of Eavg, 3-8 and Eavg, 3-36 for different cell population ratios are shown in FIGS. 9A-9D, with symbols denoting Grade 3 CD4 adverse event (AE) status (CD4+ lymphocyte decrease) following placebo or study drug treatment: Eavg, 3-36 of Treg : CD4+ Tcm ratio (FIG. 9A); Eavg, 3-36 of Treg : CD4+ Tem ratio (FIG. 9B); Eavg, 3-36 of Treg : CD8+ Tcm ratio (FIG. 9C); Eavg, 3-36 of Treg : CD8+ Tem ratio (FIG. 9D). Three out of five participants treated with 30 mg LF-vl via IM route and two out of five participants treated with 3 mg LF-vl via IV route had Grade 3 CD4+lymphocytes decrease AEs reported. As expected, those participants were associated with lower Eavg, 3-8 andEavg, 3-36 (generally at least 60% decrease from baseline). One of the participants, ID 035 of the 3 mg LF-vl IV had predose CD4+ cell counts that were less than the lower limit of the normal range. Their Grade 3 CD4+ lymphocytes decrease AE could be due to their low CD4+ cell count pretreatment despite of having Eavg, 3-8 and Eavg, 3-36 that were comparable to other participants without Grade 3 AE of CD4+ lymphocytes decrease in the same group if not higher.

[0247] FIGS. 10B-10C show the percentage of depletion of each T cell subset (as measured by AUC) between i) Hours 2 and 48 (i.e., redistribution phase; see FIG. 10A), ii) Hours 48 and 168 (i.e., early PD phase; see FIG. 10A), and iii) Hours 168 and 840 (i.e., late PD phase; see FIG. 10A). The T cell depletion results shown in FIGS. 10B-10C demonstrate durable PD in Cohorts 1-4, whereas Cohort 5 showed a large initial decrease of CD4+ Tem(FIG. 10B) and CD4+ Tcm(FIG. 10C) cells during redistribution period that was reversed by Hour 840 (i.e., Day 8) and indicative of a lack of durable PD in Cohort 5. Overall, Days 32, 42, 50, and 72 were determined to be potential targets for timing of Treg cell therapy administration.

[0248] FIGS. 11A-11B shows Treg / TSubset ratios for CD4+Tcmand CD4+Temover time. Compared to baseline, the cell subset ratios provide a good view of the variability of the lymphocytes over time. A positive ratio indicated that the Treghave increased in relative frequency compared to the denominator subset, CD4+Temor CD4+Tcm.

[0249] FIG. 12A shows baseline CD2 levels of lymphocyte subsets of the subjects in Cohorts 1-4. The baseline CD2 level varied between the subjects. FIG. 12B depicts correlation plots of the T cell depletion over a 3-36-day period (represented as average percentage change from baseline of ratios (D3-D36)) versus baseline CD2 for Cohorts 1-5.92MOFO-360045222Docket No.: 237752001540The following T cell subsets were evaluated: central memory T cells (CD4+Tcmand CD8+Tcm), effector memory T cells (CD4+Tem, CD8+Tem), including effector memory T cells reexpressing CD45RA (CD8+Temra), naive T cells (CD4+Tnand CD8+Tn), and regulatory T cells (Treg). The larger points and lines represent the predicted % depletion and its 95% confidence index (CI) associated with the mean baseline CD2 level of a T cell subset. The plots reflect the correlation between the CD2 expression level and the depletion of the T cell subsets. FIG. 12B shows that all measured cell subsets correlated with their independent maximum depletion levels. For instance, the CD4+Temsubset, which is the highest CD2 expressing subset, experienced the greatest depletion at most doses relative to the other T cell subsets. The 1 mg, 10 mg, and 30 mg intramuscular doses met the desired correlation levels. Overall, the data showed that the baseline CD2 level generally defines the relative depletion potential of LF-vl. Furthermore, sustained depletion of certain cell types (e.g., CD4+ Temand CD4+ Tem cells) can serve as a biomarker for determining the timing of Treg cell therapy administration following preconditioning with a CD-2 binding molecule of the disclosure.

[0250] FIGS. 12C-12D depict additional representations of CD2 baseline levels. Large variability in CD2 baseline levels across different T / NK cell populations was observed (FIG. 12C). Among the measured cell populations, CD4+Temcells exhibited the highest CD2 expression at baseline, while NK cells had the lowest. CD2 expression varied between individuals within the same cell type suggesting that in this healthy population the variability was normal immune heterogeneity. The relationship between CD2 baseline levels and Eavg, 3- 36 in different T / NK cell populations by treatment was explored graphically in FIG. 12D. An inverse relationship between CD2 baseline levels and Eavg, 3-36 was detected in all groups except the 3 mg IM group, suggesting larger PD responses were associated with higher CD2 baseline levels.

[0251] FIG 13 shows the time under the depletion threshold (days) at 40% and 80%. The thresholds of 40% and 80% were set for the purpose of calculating the cumulative amount of time CD4+ Temcells spent underneath the given threshold after a specific dose. This analysis is particularly useful for understanding the longevity of PD effect after a single dose. The sustained PD effect was dose-dependent.Conclusions93MOFO-360045222Docket No.: 237752001540

[0252] The objectives of this PD analysis were to assess the PD activity of the study drug after single dose administration in healthy participants and to evaluate the relationship between PK and PD on select PD endpoints.

[0253] Overall, decreases in all cell populations were observed in participants treated with an active dose of LF-vl, although the rate to nadir, the nadir and the return to baseline rate were different depending on the cell population.

[0254] Following 1 mg to 30 mg IM administration of LF-vl, dose-dependent decrease in PCFB for the absolute cell counts were observed in CD3+T cells, CD4+T cells, CD4+Tcmcells, CD4+Tem cells, CD8+T cells, CD8+Tcmcells, and CD8+Temcells. In those T cell subpopulations, maximum mean suppression up to -75% from baseline was observed around Day 2 at the 1 mg and 3 mg dose levels and within 8 hours postdose at the 10 mg and 30 mg dose levels. At the 10 mg dose, the absolute cell counts of CD4+Tcmcells, CD4+Temcells, CD8+Tcmcells, and CD8+Temcells remained suppressed at around -50% by Day 72. At the 30 mg IM dose level, absolute cell counts in the of CD3+T cell, CD4+T cell, CD4+Tcmcell, CD4+Tem cell, CD8+T cell, CD8+Tcmcell, and CD8+Temcell populations remained suppressed throughout the study, with PCFB staying below -50% until the end of the collection period on Day 72.

[0255] Following 3 mg IV administration of LF-vl, a decrease in absolute cell counts was also observed in CD3+T cells, CD4+T cells, CD4+Tcmcells, CD4+Temcells, CD8+T cells, CD8+Tcm cells, and CD8+Temcells. Maximum mean suppression was reached faster (at 2 hours postdose) and at similar magnitudes compared to that observed in the 30 mg IM group. Unlike the 30 mg IM group, PCFB following IV administration began increasing after Day 3 and was within 10% of baseline by Day 72.

[0256] Mean Treg PCFB cell counts decreased after LF-vl treatment. Mean postdose PCFB in the placebo group ranged between -31.5% and 11.7%, while mean postdose PCFB in the LF-vl between -74.1% and 26.2%. Due to the large between-subject variability in response, no clear trends could be established between dose and response in the l-10mg IM groups. At the 30mg IM dose level, Treg cells were suppressed throughout the study, with PCFB remaining around -50% by Day 72.

[0257] In CD4+Tnaive, and CD8+Tnaive cell populations, minimal to no change from baseline was observed at the 1 mg IM dose level. In higher dose groups (3 to 30 mg IM and 3 mg IV), a decrease from baseline in absolute cells counts was observed, with maximum suppression94MOFO-360045222Docket No.: 237752001540 occurring within 3 to 4 days of dosing. Tnaive cell populations remained suppressed until the end of the study in the 30 mg IM dose groups, however in the 10 mg IM group CD4+Tnaive cell counts returned to near baseline levels by Day 15.

[0258] In the CD8 Temracells and NK cells, following 1 mg to 30 mg of IM administration of LF-vl, maximum decrease in mean cell counts was observed between 8 to 24 hours postdose. For these two cell populations, the PCFB of absolute cell counts was significantly reduced by the end of the observation period post IM treatment. Following 3 mg of IV administration of LF-vl, the decrease in mean cell counts was transient, and the maximum decrease was observed within 2 hours postdose.

[0259] In the B cell population, minimal to no decrease in PCFB was observed at the 1 mg and 3 mg IM dose level. In higher dose groups (10 to 30 mg IM and 3 mg IV), a decrease from baseline in absolute cells counts was observed, with maximum suppression occurring around 8 hours and 2 hours postdose for IM and IV groups, respectively. Mean PCFB of cell counts returned to near baseline levels (>-25%) by Day 72 in all groups.

[0260] Overall, the Tregover Tcmratios for CD4+and CD8+T cells decreased compared to baseline in the 1 mg IM group, whereas for the 10 mg and 30 mg IM and 3 mg IV groups the Tregvs Tcm ratio increased compared to baseline. By the end of the observation period, mean PCFB of Treg : CD4+Tcmratios and Treg: CD8+Tcmratios ranged between -5.6% and 125.7% and between 84.5% and 222.7% for the 3 mg to 30 mg IM and 3 mg IV LF-vl-treated groups, respectively.

[0261] Overall, the Tregover Temratios for CD4+and CD8+T cells increased compared to baseline. By the end of the observation period, mean PCFB of Treg: CD4+Temratios and Treg: CD8+Tem ratios ranged between -15.4% and 444.0% and between -1.0% and 154.1% for the 3 mg to 30 mg IM and 3 mg IV LF-vl-treated groups, respectively.

[0262] Over the period of 72 days, ratios oscillated above and below 0 for the placebo group, suggesting dynamic shifts between Treg, Tcm, and Temcells.

[0263] Three out of five participants treated with 30 mg LF-vl via IM route and two out of five participants treated with 3 mg LF-vl via IV route had Grade 3 CD4+lymphocytes decrease AEs reported.

[0264] As expected, participants with Grade 3 CD4+lymphocytes decrease AE were associated with lower Eavg, 3-8 and Eavg, 3-36, except in Participant 035 of the 3 mg LF-vl IV who had predose CD4+cell counts that were less than the lower limit of the normal range.95MOFO-360045222Docket No.: 237752001540For Participant 035 Eavg, 3-8 and Eavg, 3-36 were actually comparable to other participants without Grade 3 AE of CD4+lymphocytes decrease in the same group if not higher.

[0265] An inverse relationship between CD2 baseline levels and Eavg, 3-36 was detected in all groups except the 3 mg IM group, suggesting larger PD responses were associated with higher CD2 baseline levels.

[0266] In general, higher LF-vl PK exposures were associated with greater suppression of T cells, NK cells and B cells, although R2values of the regression line were generally low (<0.5), and the strength of correlations varied among different cell populations. The correlation between PK exposures and PD responses were particularly weak for Tregcells, CD8+Tnaive cells, and B cells.

[0267] Higher LF-vl PK exposures were also associated with higher Treg: Tcmratios and Treg: Tem ratios, although R2values of the regression line were generally low (<0.30).

[0268] The participants with Grade 3 CD4+AEs had similar PK exposures to those without AEs, but markedly lower Eavg, 3-36, suggesting that AEs were not driven by high PK exposures.Example 2: In Vitro Drug-Drug Interaction (DDI) Studies

[0269] This Example assesses the drug-drug interactions (DDI) between SOI CAR Treg cells, an exemplary Treg cell therapy of the disclosure, and LF-vl. Administration of LF-vl to an individual can be used to reduce levels of highly activated effector T cells (Tetr) that are dominant in autoimmune and inflammatory disease, thus preconditioning the individual for treatment with a Treg cell therapy. However, LF-vl can also result in depletion of the Treg cell therapy itself. Accordingly, the goal of this Example was to determine serum concentrations of the CD-2 binding molecule compatible with Treg cell therapy infusion.Methods

[0270] PBMCs from 4 different autologous donors were co-cultured with CFSE-labeled SOI CAR Treg cells at effector to target ratios (E:T; PBMGSOl CAR Treg) of 25 and 5. LF- vl was also added to the co-culture at increasing concentrations (or in the absence of LF-vl; shown as far left data points at 0.05 ng / mL due to log scale) and the relative cell counts of SOI CAR Treg cells and CD4+ Temcells were determined compared to numbers in the absence of LF-vl after one day of co-culture.Results96MOFO-360045222Docket No.: 237752001540

[0271] FIGS. 14A-14D shows the LF-vl dose response of SOI CAR Treg cells and CD4+ Temcells. A maximum depletion of approximately -50% of SOI CAR Treg cells was observed. Absolute EC20 values were calculated from the curve fit analysis as the level of LF-vl corresponding to a 20% reduction in cell numbers compared to cell numbers in the absence of LF-vl. (EC20) for SOI CAR Treg cells, the median response was observed at 19 ng / mL LF-vl (PBMC:S01 = 25) and 38 ng / mL LF-vl (PBMC:S01 = 5). At EC20 for CD4+ Tem cells, the median response was observed at 50 ng / mL LF-vl (PBMC:S01 = 25) and 116 ng / mL LF-vl (PBMC:S01 = 5).Example 3: Pharmacokinetic and Pharmacodynamic Modeling

[0272] This Example provides pharmacokinetic (PK) and pharmacodynamic (PD) modeling experiments to determine the appropriate timing of Treg cell administration after preconditioning with an exemplary CD-2 binding molecule, LF-vl. Modeling experiments were based on the results of the randomized, placebo-controlled Phase I study described in Example 1 as well as the in vitro DDI studies of Example 2. Two criteria were considered for determining the timing of Treg cell administration following preconditioning with LF-vl : 1) LF-vl serum concentration (e.g., a serum concentration less than 20 ng / mL, a concentration expected to minimally impact survival of the administered Treg cells) and 2) percent T cell subset depletion (e.g., at least 40% sustained depletion of CD4+ Temor CD4+ Tcmcells, a PD response anticipated to provide a clinical benefit in autoimmune diseases) (FIG. 15).Methods

[0273] Using the pharmacokinetic data and associated Tcmeffects in cohorts 1, 3, and 4, the predicted PK and PD curves were generated. Five doses were simulated: 1, 3, 5, 7 and 10 mg. For each dose, PK concentrations from 18 subjects (intramuscular injection (IM): 1, 10, 30 mg) in Example 1 were used for the prediction. For each of the 18 subjects, 100 simulations were generated by adding variability to PK concentrations and CD4+ Tcmcounts. Thus, there were 1,800 simulated PK and PD curves for each dose.Results

[0274] FIGs. 16A-16B show the observed (FIG. 16A) and predicted (FIG. 16B) percentage change from baseline of CD4+ Tcmcells over time across multiple doses of LF- vl. The predicted percentage change from baseline (%CFB) of CD4+ Tcmcells at Days 32, 42, and 56 is provided in Table 8 below. Median estimates were obtained by computing the median of the 100 medians, each summarizing N=18 simulated subjects. 90% CI of the97MOFO-360045222Docket No.: 237752001540 median was obtained by computing the [5th%, 95th%] of the 100 medians. 90% PI (prediction interval) was obtained as medians of the 100 values, each representing 5th% and 95th% of values for N=18 simulated subjects.*% subjects with CD4Tcm %CFB < -25% or -50%

[0275] FIGS. 17A-17B show the observed (FIG. 17A) and predicted (FIG. 17B) pharmacokinetic profiles across multiple doses of LF-vl. The projected days to reach <20 ng / mL, <40 ng / mL, and <100 ng / mL serum concentration of LF-vl for 25%, 50% and 75% of the subjects are provided in Table 9 below. For a 5 mg dose of LF-vl, the median treatment days when 50% of subjects have <100 ng / mL serum concentration of LF-vl was 40.8 days. The median treatment days when 75% of subjects have <100 ng / mL serum concentration of LF-vl was 48.9 days.98MOFO-360045222Docket No.: 237752001540

[0276] Taking into consideration the PK and PD curves for each dose, a matrix was created describing PK and PD considerations (D32 PD (Tcm depletion), D32 / D42 PK (ng / mL), Day PK <20 ng / mL) that can be considered to determine the timing of Treg cell administration following preconditioning with LF-vl (Table 10). For example, the modeling and observed clinical data indicates that a 5 to 10 mg IM dose would be associated with > 40% sustained depletion of CD4+ Tcmcells for up to 72 days and serum levels of LF-vl of less than 20 ng / mL in approximately 80 to 90 days post-dose. Accordingly, 80 to 90 days after preconditioning with a 5 to 10 mg dose of LF-vl could provide an appropriate time window for administration of a Treg cell therapy.

[0277] The present disclosure is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the disclosure. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.99MOFO-360045222

Claims

1. Docket No.: 237752001540CLAIMSWhat is claimed is:

1. A method of treating an autoimmune disease or inflammatory disease in an individual comprising administering to the individual a) about 1 mg to about 30 mg of a CD-2 binding molecule comprising a CD2-binding domain of human leukocyte function-associated antigen 3 (LFA3) linked to an Fc domain; and b) a composition comprising modified regulatory T (Treg) cells, wherein the modified Treg cells express a therapeutic protein; wherein the composition comprising the modified Treg cells is administered after administration of the CD-2 binding molecule.

2. The method of claim 1, wherein about 3 mg to about 10 mg of the CD-2 binding molecule is administered to the individual.

3. The method of claim 1 or 2, wherein about 3 mg of the CD-2 binding molecule is administered to the individual.

4. The method of claim 1 or 2, wherein about 5 mg of the CD-2 binding molecule is administered to the individual.

5. The method of claim 1 or 2, wherein about 7 mg of the CD-2 binding molecule is administered to the individual.

6. The method of claim 1 or 2, wherein about 10 mg of the CD-2 binding molecule is administered to the individual.

7. The method of any one of claims 1-6, wherein the CD-2 binding molecule is administered by intramuscular injection.

8. The method of any one of claims 1-7, wherein about 3 mg, 5 mg, 7 mg, or 10 mg of the CD-2 binding molecule is administered to the individual, and the CD-2 binding molecule is administered by intramuscular injection.100MOFO-360045222Docket No.: 2377520015409. The method of any one of claims 1-6, wherein the CD-2 binding molecule is administered intravenously.

10. The method of any one of claims 1-3 and 9, wherein about 3 mg of the CD-2 binding molecule is administered to the individual and the CD-2 binding molecule is administered intravenously.

11. The method of any one of claims 1-10, wherein the LFA3 domain comprises an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 3.

12. The method of any one of claims 1-11, wherein the LFA3 domain comprises the amino acid sequence of SEQ ID NO: 3.

13. The method of any one of claims 1-12, wherein the Fc domain comprises an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 4.

14. The method of any one of claims 1-13, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO: 4.

15. The method of any one of claims 1-14, wherein the CD-2 binding molecule comprises an amino acid sequence with at least about 90% identity to the amino acid sequence of SEQ ID NO: 2.

16. The method of any one of claims 1-15, wherein the CD-2 binding molecule comprises the amino acid sequence of SEQ ID NO: 2.

17. The method of any one of claims 1-16, wherein the composition comprising the modified Treg cells is administered about 50 days to about 120 days after administration of the CD-2 binding molecule.101MOFO-360045222Docket No.: 23775200154018. The method of any one of claims 1-17, wherein the composition comprising the modified Treg cells is administered about 80 days to about 90 days after administration of the CD-2 binding molecule.

19. The method of any one of claims 1-18, wherein administration of the CD-2 binding molecule to the individual results in a reduction of CD4+ effector memory T-cells (Tern) in the peripheral blood of the individual.

20. The method of claim 19, wherein the composition comprising the modified Treg cells is administered when the level of CD4+ Tern in the peripheral blood of the individual is reduced by at least about 40% compared to the level of CD4+ Tern in the peripheral blood of the individual prior to administration of the CD-2 binding molecule.

21. The method of claim 19, wherein the composition comprising the modified Treg cells is administered when the level of CD4+ Tern in the peripheral blood of the individual is reduced by about 25% to about 50% compared to the level of CD4+ Tern in the peripheral blood of the individual prior to administration of the CD-2 binding molecule.

22. The method of any one of claims 1-21, wherein administration of the CD-2 binding molecule to the individual results in a reduction of CD4+ central memory T-cells (Tcm) in the peripheral blood of the individual.

23. The method of claim 22, wherein the composition comprising the modified Treg cells is administered when the level of CD4+ Tcm in the peripheral blood of the individual is reduced by at least about 40% compared to the level of CD4+ Tcm in the peripheral blood of the individual prior to administration of the CD-2 binding molecule.

24. The method of claim 22, wherein the composition comprising the modified Treg cells is administered when the level of CD4+ Tcm in the peripheral blood of the individual is reduced by about 30% to about 50% compared to the level of CD4+ Tcm in the peripheral blood of the individual prior to administration of the CD-2 binding molecule.102MOFO-360045222Docket No.: 23775200154025. The method of any one of claims 1-24, wherein the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the serum of the individual is less than about 50 ng / mL.

26. The method of any one of claims 1-25, wherein the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the serum of the individual is less than about 20 ng / mL.

27. The method of any one of claims 1-24, wherein the composition comprising the modified Treg cells is administered when the concentration of the CD-2 binding molecule in the serum of the individual is about 10 ng / mL to about 50 ng / mL.

28. The method of any one of claims 1-27, wherein the composition comprising the modified Treg cells is administered intravenously.

29. The method of any one of claims 1-3, 7, 8, 11-17, and 19-28, wherein about 3 mg of the CD-2 binding molecule is administered to the individual, wherein the CD-2 binding molecule is administered by intramuscular injection, and wherein the composition comprising the modified Treg cells is administered about 65 days to about 80 days after administration of the CD-2 binding molecule.

30. The method of any one of claims 1-2, 4, 7, 8, 11-17, and 19-28, wherein about 5 mg of the CD-2 binding molecule is administered to the individual, wherein the CD-2 binding molecule is administered by intramuscular injection, and wherein the composition comprising the modified Treg cells is administered about 75 days to about 95 days after administration of the CD-2 binding molecule.

31. The method of any one of claims 1-2, 5, 7, 8, 11-17, and 19-28, wherein about 7 mg of the CD-2 binding molecule is administered to the individual, wherein the CD-2 binding molecule is administered by intramuscular injection, and wherein the composition comprising the modified Treg cells is administered about 80 days to about 105 days after administration of the CD-2 binding molecule.103MOFO-360045222Docket No.: 23775200154032. The method of any one of claims 1-2, 6, 7, 8, 11-17, and 19-28, wherein about 10 mg of the CD-2 binding molecule is administered to the individual, wherein the CD-2 binding molecule is administered by intramuscular injection, and wherein the composition comprising the modified Treg cells is administered about 85 days to about 120 days after administration of the CD-2 binding molecule.

33. The method of any one of claims 1-3, 9-17, and 19-28, wherein about 3 mg of the CD-2 binding molecule is administered to the individual, wherein the CD-2 binding molecule is administered intravenously, and wherein the composition comprising the modified Treg cells is administered about 55 days to about 80 days after administration of the CD-2 binding molecule.

34. The method of any one of claims 1-33, wherein the therapeutic protein is a T cell receptor (TCR) comprising an antigen-binding site that specifically binds to antigen.

35. The method of any one of claims 1-33, wherein the therapeutic protein is a chimeric antigen receptor (CAR).

36. The method of any one of claims 1-35, wherein the modified Treg cells are CD4+, CD25+ and CD1271o.

37. The method of claim 35, wherein the CAR comprises an antigen binding domain that specifically binds to an antigen that is associated with an autoimmune disease or inflammatory disease.

38. The method of claim 37, wherein the antigen is selected from the group consisting of MHC class I, MHC class II, collagen type V, K alphal tubulin, MHC class I related chain A (MICA), fibronectin, collagen type IV, collagen type VI, vimentin, angiotensin II type 1 receptor (AGTR1), perlecan, agrin, collagen type I, collagen type II, collagen type III, cardiac myosin, an islet cell autoantibody (ICA) antigen, insulin, glutamic acid decarboxylase (GAD), myelin, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, astrocyte proteins, glial fibrillary protein (GFAP), SlOObeta, beta cell antigen, insulin B chain, proinsulin, preproinsulin, glutamic acid decarboxylase-65 (GAD65), islet- associated antigen 2, islet-specific glucose-6-phosphatase catalytic subunit-related protein104MOFO-360045222Docket No.: 237752001540(IGRP), zinc transporter 8 (ZnT8), islet antigen 2 (IA-2), heat shock protein 60 (HSP60), chromogranin A, dnaJ (heat shock protein), citrullinated-vimentin, human cartilage glycoprotein-39, mitochondrial component, pyruvate dehydrogenase (mitochondrial), E2 component of pyruvate dehydrogenase, E2 component of branched chain 2-oxo acid dehydrogenase, E2 component of 2-oxo-glutarate dehydrogenase complex, E3 binding protein of dihydrolipoamide dehydrogenase, a nuclear component, nuclear protein splOO, nuclear pore complex protein gpl20, centromere, acetylcholine receptor (AChR), aquaporin-4 (AQP-4), CTLA-4, ICAM, LFA-3, CD40 / CD154, ICOS / ICOSL, CD52, nuclear factor of activated T cells (NF AT), phospholipase C (PLC), CD25, Janus kinase, B cell activating factor (BAFF), a proliferating inducing ligand (APRIL), IL6R, IL 17, IL12 / IL23, an integrin, a sphingosin receptor, melanocyte antigen, toll-like receptor, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, MyD88, an IL-lR-associated kinase (IRAK), an allergen associated with the allergic disorder, a citrullinated protein or citrullinated fragments thereof, citrullinated vimentin, and citrullinated filaggrin.

39. The method of claim 37 or 38, wherein the antigen binding domain specifically binds to one or more different citrullinated proteins or citrullinated fragments thereof.

40. The method of any one of claims 37-39, wherein the antigen binding domain of the CAR specifically binds to citrullinated vimentin.

41. The method of any one of claims 37-40, wherein the antigen binding domain of the CAR binds to all three of (i) citrullinated vimentin, (ii) citrullinated filaggrin, and (iii) citrullinated fibrinogen, or citrullinated peptides fragments thereof, optionally wherein the antigen binding domains further binds to citrullinated tenascin C.

42. The method of any one of claims 1-41, wherein the modified Treg cells are human T cells.

43. The method of any one of claims 1-42, wherein the modified Treg cells are primary T cells.

44. The method of any one of claims 1-43, wherein the individual is a human.105MOFO-360045222Docket No.: 23775200154045. The method of any one of claims 1-44, wherein the autoimmune disease or inflammatory disease is a disease mediated by CD2.

46. The method of any one of claims 1-45, wherein the autoimmune disease or inflammatory disease is selected from the group consisting of type I diabetes, rheumatoid arthritis, hidradenitis suppurativa, amyotrophic lateral sclerosis, celiac disease, and inflammatory bowel disease.106MOFO-360045222

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