ANTI-CANCER COMBINATION THERAPIES COMPRISING CTLA-4 AND PD-1

BLOCKING AGENTS

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to anti-cancer combination therapies comprising a CTLA-4 blocking agent and a PD-1 blocking agent. In particular, the present invention relates to combination therapies wherein the CTLA-4 blocking agent is an anti-CTLA-4 antibody with reduced or no measurable effector function or an anti-CTLA-4 antibody fragment that lacks an Fc domain, and the PD-1 blocking agent is an anti-PD-1 antibody, anti-PD-1 antibody fragment, anti-PD-Ll antibody, or anti-PD-Ll antibody fragment.

(2) Description of Related Art

Tumor immunotherapy has assumed a more prominent role for treatment of a variety of cancer indications. The clinical successes utilizing antibody blockade of immune checkpoint inhibitory receptors expressed on T cells such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death receptor- 1 (PD-1) has galvanized the notable advancement of immunotherapy for cancer. CTLA-4 or PD-1 monotherapy blockade by monoclonal antibodies (mAbs) has resulted in enhanced anti-tumor responses and beneficial clinical outcomes in controlled randomized clinical trials.

A prominent feature of the immune checkpoint blockade for treating various cancers is the clinically validated benefit of combination therapies that include anti-PD-1 and anti-CTLA-4 antibodies. As more and more clinical data is released, it is becoming clear that anti-PD-1 /CTLA-4 combination therapies may provide superior clinical efficacy when compared to targeting either checkpoint pathway alone. However, immune-related toxicities (irAEs) associated with anti-CTLA-4 antibodies have been significant in both monotherapy settings and in combination therapies with anti-PD-1 antibodies. For example, the anti-CTLA-4 antibody ipilimumab, which is marketed by Bristol-Myers Squibb under the tradename YERVOY and is the only anti-CTLA-4 antibody approved by the United States Food and Drug Administration (U.S. FDA), is subject to a Black Box warning due to its potential to induce severe and fatal immune-mediated adverse reactions such as inflammation of the intestines, liver, skin, hormone- producing glands, and/or eyes. Ipilimumab has also been approved for combination therapies with the anti-PD-1 antibody nivolumab (marketed by Bristol-Myers Squibb under the tradename OPDIVO) for advanced renal cell carcinoma and certain colorectal cancers but due to the risk for significant irAEs, ipilimumab is administered at a low or subtherapeutic dose of 1 mg/kg. The subtherapeutic dose for the combination therapy is significantly lower than the 3 mg/kg monotherapy dose for unresectable or metastatic melanoma or the 10 mg/kg monotherapy dose for adjuvant melanoma (see Package Insert and Label for YERVOY (July 2018)).

Both FDA-approved anti -PD 1 mAbs, nivolumab and pembrolizumab, are humanized anti-PDl IgG4 kappa antibodies, which are disclosed in U.S patent No. 8,008,449 and U.S. Pat. No. 8,354,509, respectively. The IgG4 isotype Fc domain is generally recognized as having little detectable effector function.

Ipilimumab is a human anti-CTLA-4 IgG _] kappa antibody, which is disclosed in U.S. Pat. No. 6,984,720. The heavy chain (HC) constant domain of the IgG _j isotype has an Fc domain that is generally recognized as having high affinity for Fc receptors (FcR), which provides significant effector function to the antibody (e.g., inducing antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or Complement- dependent cytotoxicity (CDC)). Research has shown that Fc effector function is required for efficacy of anti-CTLA-4 antibodies. For example, Ingram et al, Proc. Natl. Acad. Sci. USA 115: 3912-3917 (2018) showed in a mouse model that an anti-CTLA-4 alpaca heavy chain-only antibody fragment (VHH) that lacks a heavy chain Fc domain and its attendant effector function had no anti-tumor efficacy; however, anti-tumor efficacy could be restored to the molecule by fusing it to a mouse IgG2 heavy chain Fc domain displaying effector function; and, Selby et al, Cancer Immunol. Res. 1 : 32-42 (2013) showed in a mouse model that anti-CTLA-4 antibodies fused or linked to an Fc domain mutated to eliminate effector function did not display any anti tumor activity. See also Simpson et al, J. Exp. Med.;210: 1695-710 (2013) and International Patent Application No. W02014089113.

Tremelizumab is a human anti-CTLA-4 human IgG2 antibody, which has been disclosed in U.S. Pat. No. 8,491,895. The human IgG2 isotype had been selected to minimize potential effector function activity and thereby potentially reduce irAEs. However, as shown in Vargas et al, Cancer Cell 33: 649-663 (2018), tremelizumab retains effector function; and Bertrand et al, BMC Med. 13: 211-214 (2015) showed that while tremelizumab could be administered at a dose higher than that for ipilimumab, it was still capable of inducing irAEs, in particular gut and skin inflammatory immune-mediated toxi cities. See also Ribas et al.,The Oncologist 12: 873-993 (2007), Schneider-Merck et al, J. Immunol. 184: 512-520 (2010) and Konitzer et al, PLoS One. 10:e0145633(2015), which showed other human IgG2 antibodies that induce ADCC and ADCP in vitro of similar equivalence to the human IgG _j isoform.

Other attempts to reduce irAEs of ipilimumab include BMS-986249, a probody composed of ipilimumab linked to a proprietary masking peptide that covers the active antigen binding site of the antibody through a protease-cleavable linker. The masking peptide may reduce irAEs by minimizing ipilimumab’ s ability to bind CTLA-4 in normal tissues (See, International patent Applications W02009025846, W02010081173, WO2018222949, WO2018085555, Pai et al., J. Clin. Invest. 129: 349-363 (2019), and Korman et al., Abstract SY09-01, AACR Annual Meeting Vol 77, issue 13 (2017)).

In light of studies suggesting that the therapeutic efficacy of anti-CTLA-4 antibodies like ipilimumab may involve depletion of regulatory T cells (T _re g _S ), it has been proposed that anti-CTLA-4 antibodies, such as ipilimumab, that have enhanced ADCC activity would provide more effective anti-tumor activity than current antibodies. U.S. Pat. No.

10,196,445 discloses several ipilimumab variants with enhanced ADCC activity.

The standard-of-care for some anti-cancer therapies comprises providing an anti- PD-1 antibody in combination with chemotherapy. The anti -tumor activity of an anti-CTLA-4 antibody may further enhance the efficacy of these therapies; however, because gastrointestinal toxicity is one of the most commonly encountered side effects experienced during chemotherapy, addition of an anti-CTLA-4 antibody to the therapy may instead exacerbate the gastrointestinal toxicity.

Clearly, anti- CTLA-4 antibodies that enabled dosing at higher, more optimal levels, without associated irAEs, in particular the skin and gut inflammatory immune-mediated toxicities associated with current anti-CTLA-4 antibodies, would likely allow for more effective therapies in combination with anti-PD-1 antagonists and, optionally, chemotherapies.

BRIEF SUMMARY OF THE INVENTION

The inventors have discovered that while certain CTLA-4 blocking agents that bind CTLA-4 have reduced or no measurable anti-tumor activity when administered as a monotherapy, they may display clinically relevant anti-tumor activity when used in combination therapies with a PD-1 blocking agent. The inventors have also discovered that these certain CTLA-4 blocking agents may exert anh -tumor activity in a CTLA-4/PD-1 blockade combination therapy without inducing the immune-mediated adverse reactions (irAEs), including the irAEs in the skin and gut, that have been associated with the currently approved CTLA-4/PD-1 blockade combination therapies. The CTLA-4/PD-1 blockade combinations disclosed herein enable therapies of increased therapeutic index over the current CTLA-4/ PD-1 blockade combination therapies including combination therapies that include chemotherapy, which may lead to more efficacious cancer treatments with improved patient outcomes.

The certain CTLA-4 blocking agents used as part of CTLA-4/PD-1 blockade combination therapy of the present invention may be selected from the group consisting of (i) an effector-silent anti-CTLA-4 antibody and (ii) an effector-silent anti-CTLA-4 antibody fragment that either lacks a fragment crystallizable (Fc) domain or has an Fc domain that comprises deletions of those regions in the Fc domain that bind the Fc receptors (FcRs). An effector-silent antibody or antibody fragment displays either (i) no measurable binding to one or more FcRs, as may be measured in a Biacore assay wherein an association constant in the micromolar range indicates no measurable binding or (ii) measurable binding to one or more FcRs as may be measured in a Biacore assay that is reduced compared to the binding that is typical for an antibody of the same isotype. These certain CTLA-4 blocking agents are effector-silent CTLA-4 blocking agents.

In particular embodiments, these effector-silent anti-CTLA-4 antibodies and effector-silent anti-CTLA-4 antibody fragments may not display measurable anti-tumor activity in an anti-cancer monotherapy but will display measurable anti-tumor activity in a combination anti-cancer therapy with a PD-1 or PD-L1 blocking agent and without displaying the irAEs typically associated with CTLA-4/PD-1 blockade combination therapies, in particular skin or gut inflammatory immune-related toxicities.

The effector-silent anti-CTLA-4 antibodies or effector-silent anti-CTLA-4 antibody fragments disclosed herein may be used at higher doses and for longer time periods in combination with PD-1 or PD-L1 blocking agents without displaying the irAEs typically associated with CTLA-4/PD-1 blockade combination therapies, in particular skin or gut inflammatory immune-related toxicities. Currently, the anti-CTLA-4 antibody ipilimumab dose approved for use in anti-CTLA-4/PD-l blockade combination therapies is 1 mg/kg compared to the 3 mg/kg or 10 mg/kg dose approved for use in monotherapies (See Package Insert and Label for YERVOY (July 2018)) or the 100 mg or less fixed dose contemplated for CTLA-4/PD-1 blockade combination therapies in International Patent Application WO2018183408. Thus, the CTLA-4/PD-1 blockade combination therapies of the present invention may use effector-silent anti-CTLA-4 antibodies or effector-silent anti-CTLA-4 antibody fragments at doses that are the same as or higher than the doses currently approved for anti-CTLA-4 antibodies in

monotherapies. The effector-silent anti-CTLA-4 antibodies or anti-CTLA-4 antibody fragments disclosed herein may also be used in combination with anti-PD-1 or anti-PD-Ll antibodies at doses similar to those currently used in or contemplated for CTLA-4/PD-1 blockade combination therapies but for a longer duration of time than is currently obtainable for anti-CTLA-4 antibodies and without displaying the irAEs typically associated with CTLA-4/PD-1 blockade combination therapies, in particular skin or gut inflammatory immune-related toxicities.

Accordingly, the present invention provides a combination therapy for treating cancer in an individual in need of such treatment, the method comprising administering to an individual with a cancer (i) a therapeutic dose of a PD-1 or PD-L1 blocking agent and (ii) a therapeutic dose of an effector-silent CTLA-4 blocking agent, to treat the cancer, wherein the effector-silent CTLA-4 blocking agent displays anti-tumor activity in the combination therapy that it does not display when administered to an individual in a monotherapy without the PD-1 or PD-L1 blocking agent. In further embodiments, the combination therapy does not induce or has reduced risk of inducing immune-mediated adverse reactions (irAEs) in the gut or skin during the course of the combination therapy that is greater than Grade 2 as defined in Common Terminology Criteria for Adverse events (CTCAE) Version 5.0 compared to combination therapies comprising an anti-CTLA-4 antibody displaying effector function. In particular embodiments, the effector-silent CTLA-4 blocking agent used as part of the combination therapy does not induce irAEs in the skin or gut that is greater than Grade 2 for at least the first 10 weeks of combination therapy. In particular embodiments, the combination therapy does not result in detectable irAEs for at least the first four weeks of the combination therapy or irAE greater than Grade 1 for at least the first four weeks of the combination therapy.

In one embodiment, the effector-silent CTLA-4 blocking agent used as part of the combination therapy described herein is an effector-silent anti-CTLA-4 antibody or an effector- silent anti-CTLA-4 antibody fragment.

In another embodiment, the PD-1 blocking agent used as part of the combination therapy described herein is an anti-PD-1 or anti-PD-Ll antibody or an anti-PD-1 or an anti-PD- L1 antibody fragment. In particular embodiments, the anti-PD-1 or anti-PD-Ll antibody comprises an HC domain comprising one or more mutations in the Fc domain that render the antibody effector-silent. The PD-1 blocking agent may also be an anti-PD-1 or an anti-PD-Ll antibody fragment, each of which lacks an Fc domain or those regions of the Fc domain that bind one or more FcRs, which renders the antibody fragment effector-silent.

The present invention further provides anti-cancer combination therapies, which comprise, administering to an individual in need of a cancer therapy (i) a first formulation comprising a PD-1 blocking agent selected from the group consisting of an anti-PD-1 antibody having an IgG or IgG2 Fc domain, an effector-silent anti-PD-1 antibody, and effector-silent anti-PD-1 antibody fragment; and, (ii) a second formulation comprising an effector-silent CTLA- 4 blocking agent selected from the group consisting of an effector-silent anti-CTLA-4 antibody and an effector-silent anti-CTLA-4 antibody fragment.

The present invention further provides an anti-cancer combination therapy, which comprises administering to an individual in need of a cancer therapy a formulation comprising (i) a PD-1 blocking agent selected from the group consisting of an anti-PD-1 antibody having an IgG4 or IgG2 Fc domain, an effector-silent anti-PD-1 antibody, and effector-silent anti-PD-1 antibody fragment; and, (ii) an effector-silent CTLA-4 blocking agent selected from the group consisting of an effector-silent anti-CTLA-4 antibody and an effector-silent anti-CTLA-4 antibody fragment.

The present invention further provides anti-cancer combination therapies, which comprise, administering to an individual in need of a cancer therapy (i) a first formulation comprising a PD-L1 blocking agent selected from the group consisting of an anti-PD-Ll antibody having an IgG4 or IgG2 Fc domain, an effector-silent anti-PD-Ll antibody, and effector-silent anti-PD-Ll antibody fragment; and, (ii) a second formulation comprising an effector-silent CTLA-4 blocking agent selected from the group consisting of an effector-silent anti-CTLA-4 antibody and an effector-silent anti-CTLA-4 antibody fragment.

The present invention further provides an anti-cancer combination therapy, which comprises administering to an individual in need of a cancer therapy a formulation comprising (i) a PD-L1 blocking agent selected from the group consisting of an anti-PD-Ll antibody having an IgG4 or IgG2 Fc domain, an effector-silent anti-PD-Ll antibody, and an effector-silent anti-

PD-Ll antibody fragment; and, (ii) an effector-silent CTLA-4 blocking agent selected from the group consisting of an effector-silent anti-CTLA-4 antibody and an effector-silent anti-CTLA-4 antibody fragment.

In more specific embodiments of the combination therapy, the effector-silent anti- CTLA-4 antibody comprises an IgG _j Fc domain having (i) a mutation in the A-glycosylation site

Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes X-glycosylation at said N- glycosylation site or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; (ii) an amino acid substitution mutation selected from the group consisting ofN297A, L234A/L235A/D265A,

L234A/L235A/P329G, L235E, D265A, E233A/L235A,S267E/L328F, S2339D/A330L/I332E, L235G/G236R, N297A/D356E/L358M, L234F/L235E/P331S/D365E/L358M, and

D265A/N297G or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; or (iii) a mutation in the N- glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes N- glycosylation at said X-glycosylation site and an amino acid substitution mutation selected from the group consisting of L234A/L235A/D265A, L234A/L235A/P329G, L235E, D265A, E233A/L235A,S267E/L328F, S2339D/A330L/I332E, L235G/G236R, D356E/L358M,

L234F/L235E/P331S/D365E/L358M, and D265A or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

In particular embodiments of the combination therapy, the effector-silent anti- CTLA-4 antibody comprises an IgG2 Fc domain having (i) a mutation in the X-glycosylation site

Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes X-glycosylation at said N- glycosylation site or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; (ii) an amino acid substitution mutation selected from the group consisting ofN297A/D265S, D265A, P329G/D265A/N297G, or V234A/G237A/P238S/H268A/V309L/A330S/P331S or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; or (iii) a mutation in the A-glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes X-glycosylation at said X-glycosylation site and an amino acid substitution mutation selected from the group consisting of N297A/D265S, D265A, P329G/D265A/N297G, or V234A/G237A/P238S/H268A/V309L/A330S/P331S or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

In particular embodiments of the combination therapy, the effector-silent anti- CTLA-4 antibody comprises an IgG4 Fc domain having an S228P amino acid substitution and further comprising (i) a mutation in the V-glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes V-glycosylation at said X-glycosylation site or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; (ii) an amino acid substitution mutation selected from the group consisting of N267A, P329G, and D265A/N297A or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; or (iii) a mutation in the V-glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes X-glycosylation at said X-glycosylation site and an amino acid substitution mutation selected from the group consisting of N267A, P329G, and D265A/N297A or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

In a further embodiment of the combination therapy, the effector-silent anti- CTLA-4 antibody fragment, which lacks an Fc domain, is or comprises a single-chain variable fragment (scFv), an antigen binding fragment (Fab), or an antigen binding fragment dimer

F(ab’) ₂ ·

In particular embodiments of the combination therapy, the effector-silent anti- CTLA-4 antibody or effector-silent anti-CTLA-4 antibody fragment comprises the three heavy chain (HC) complementarity determining regions (CDRs) and three light chain (LC) CDRs of an anti-CTLA-4 antibody selected from the group consisting of ipilimumab, tremelimumab, REGN4659, AGEN1884w, 8D2/8D2 (RE), 8D2/8D2 (RE)-Variant 1, 8D2H1L1, 8D2H1L1- Variant 1, 8D2H2L2, 8D2H2L2-Variant 1, 8D3H3L3, 8D2H2L15, 8D2H2L 15 -Variant 1, 8D2H2L17, and 8D2H2L17-Variant 1.

In particular embodiments of the combination therapy, the effector-silent anti- CTLA-4 antibody or effector-silent anti-CTLA-4 antibody fragment comprises the V _[ and VL of ipilimumab, the V _[ and VL of tremelimumab, the V _[ and V of REGN4659, the V _]-[ and V _L of AGEN 1884w, the V _H and V _L of 8D2/8D2 (RE), the V _H and V _L of 8D2/8D2 (RE)- Variant 1, the V _]-[ and VL of 8D2H1L1, the V _]-[ and VL of 8D2H1 LI -Variant 1, the V _]-[ and V _L of 8D2H2L2, the V _H and V _L of 8D2H2L2-Variant l,the V _H and V _L of 8D3H3L3, the V _H and V _L of 8D2H2L15, the V _H and V _L of 8D2H2L 15-Variant 1, the V _H and V _L of

8D2H2L17, or the VH and VL of 8D2H2L17-Variant 1. In particular embodiments, the effector-silent anti-CTLA-4 antibody or effector- silent anti-CTLA-4 antibody fragment comprises (i) a V _j-[ comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 8; (ii) a V _j q comprising the amino acid sequence set forth in SEQ ID NO: 15 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 16; (iii) aVp comprising the amino acid sequence set forth in SEQ ID NO:95 and a V comprising the amino acid sequence set forth in SEQ ID NO:96; or, (iv) a V _j q having the amino acid sequence set forth in SEQ ID NO:97 and a VL having the amino acid sequence set forth in SEQ ID NO:98.

In particular embodiments, the effector-silent anti-CTLA4 antibody or effector- silent anti-CTLA-4 antibody fragment comprises (i) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:73 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:74; (ii) a VH domain comprising the amino acid sequence set forth in SEQ ID NO:75 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:76;

(iii) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:77 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:78; (iv) a Vq domain comprising the amino acid sequence set forth in SEQ ID NO:79 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 80; (v) aVp domain comprising the amino acid sequence set forth in SEQ ID NO:81 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 82; (vi) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:83 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 84; (vii) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO: 85 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:86; (viii) a Vq domain comprising the amino acid sequence set forth in SEQ ID NO:87 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 88; (ix) aVp domain comprising the amino acid sequence set forth in SEQ ID NO: 89 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:90; (x) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:91 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:92; or (xi) a Vp domain comprising the amino acid sequence set forth in SEQ ID NO:93 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:94.

In further embodiments of the combination therapy, the effector-silent CTLA-4 blocking agent is an effector-silent anti-CTLA-4 antibody selected from the effector-silent anti- CTLA-4 antibodies disclosed in Tables 4-18.

In a further embodiment of the combination therapy, the effector-silent CTLA-4 binding agent is an effector-silent anti-CTLA-4 antibody fragment that comprises one or more immunoglobulin single variable domains (ISVDs), each ISVD comprising the variable domain (VHH) °f ^a camelid heavy chain only antibody; with the proviso that none of the ISVDs comprises a VHH having a CDR1 comprising the amino sequence FYGMG (SEQ ID NO:69, a CDR2 comprising the amino acid sequence DIRTS AGRTTY AD SVKG (SEQ ID NO:70), and a CDR3 comprising amino acid EMSGISGWDY (SEQ ID NO:71) or EPSGISGWDY (SEQ ID NO:72) as those ISVDs are disclosed in International Patent Application W02008071447, WO2017087587, and WO2017087588, or a VHH that comprises 1, 2, or 3 mutations in CDR3 as disclosed in W02008071447, with the exception that ISVDs comprising said CDRs in embodiments wherein the one or more ISVDs are fused or linked to an effector-silent heterologous HC domain or Fc domain, including, for example, any one of the effector-silent antibody HC domains or Fc domains disclosed herein are not excluded by this proviso.

In particular embodiments of the combination therapy, the anti-PD-1 antibody or anti-PD-1 antibody fragment comprises the three heavy chain complementarity determining regions (CDRs) and three light chain CDRs of pembrolizumab, nivolumab, or cemiplimab-rwlc. In particular embodiments of the combination therapy, the anti-PD-1 antibody comprises (i) the V _j q and VL of pembrolizumab; (ii) the V _j q and VL of nivolumab; or, (iii) the V _j q and VL of cemiplimab-rwlc.

In further embodiments, the anti-PD-1 antibody or anti-PD-1 antibody fragment comprises (i) a V _j q having the amino acid sequence set forth in SEQ ID NO:29 and a VL having the amino acid sequence set forth in SEQ ID NO:30; (ii) a V _j q having the amino acid sequence set forth in SEQ ID NO:23 and a V having the amino acid sequence set forth in SEQ ID NO:24; or, (iii) a V _j q having the amino acid sequence set forth in SEQ ID NO:99 and a VL having the amino acid sequence set forth in SEQ ID NO: 100. In a further embodiment, the anti- PD1 antibody comprises (i) a HC having the amino acid sequence set forth in SEQ ID NO:27 and a LC having the amino acid sequence set forth in SEQ ID NO:28; (ii) an HC having the amino acid sequence set forth in SEQ ID NO:25 and a LC having the amino acid sequence set forth in SEQ ID NO:26; or (iii) an HC having the amino acid sequence set forth in SEQ ID NO: 101 and a LC having the amino acid sequence set forth in SEQ ID NO: 102.

In particular embodiments of the combination therapy, the anti-PD-Ll antibody or anti-PD-Ll antibody fragment comprises (i) the V _j q and VL domains of atezolizumab; (ii) the Vjq and VL domains of avelumab; or, (iii) the Vjq and VL domains of durvalumab.

In further embodiments; the anti-PD-Ll antibody or anti-PD-Ll antibody fragment comprise (i) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO: 103 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 104; (ii) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO: 105 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 106; or, (iii) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO: 107 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 108.

In particular embodiments of the combination therapy, the anti-PD-1 or anti-PD- Ll antibody may comprise an IgG _j , IgG2, or IgGq Fc domain as disclosed herein, which may comprise a C-terminal lysine or lack either a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

In particular embodiments of the combination therapy, the anti-PD-1 or anti-PD- L1 antibody comprises (i) an IgG2 or IgG4 Fc domain; (ii) an IgG _| . IgG2, or IgG4 Fc domain comprising a mutation in the A-glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes A-glycosylation at said A-glycosylation site or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; (iii) an IgG _j Fc domain comprising N297 A, L234A/L235A/D265A,

L234A/L235A/P329G, L235E, D265A, , E233A/L235A, L235G/G236R, S267E/L328F, S2339D/A330L/I332E, or D265A/N297G amino acid substitutions or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; (iv) an IgG2 Fc domain comprising N297A/D265S, D265A,

P329G/D265A/N297G, or V234A/G237A/P238S/H268A/V309L/A330S/P331S amino acid substitutions or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; or (v) an IgG4 Fc domain comprising an S228P amino acid substitution and an N267A, P329G, or D265A/N297A amino acid substitution or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein the amino acid positions are identified according to Eu numbering.

In particular embodiments of the combination therapy, the PD-1 blocking agent is an anti-PD-1 antibody selected from the anti-PD-1 antibodies disclosed in Tables 19-27 or an anti-PD-Ll antibody selected from the anti-PD-Ll antibodies disclosed in Tables 28-36.

In a further embodiment of the combination therapy, the anti-PD-1 antibody fragment or anti-PD-Ll antibody fragment, each of which lacks an Fc domain, is a single-chain variable fragment (scFv), an antigen binding fragment (Fab), or an antigen binding fragment dimer F(ab’)2·

In a further embodiment of the combination therapy, the anti-PD-1 or anti-PD-Ll antibody fragment comprises one or more ISVDs, each ISVD comprising the VHH °f ^a camelid heavy chain only antibody.

In particular embodiments of the combination therapy, the CTLA-4 blocking agent is administered at a dose comprising about 1 mg/kg to about 3 mg/kg of the CTLA-4 blocking agent or a fixed dose of the CTLA-4 blocking agent that does not depend on the individual’s weight and is greater than about 100 mg.

In particular embodiments of the combination therapy, the CTLA-4 blocking agent is administered at a dose comprising between 1 mg/kg and 3 mg/kg of the CTLA-4 blocking agent. In particular embodiments of the combination therapy, the CTLA-4 blocking agent is administered at a dose comprising between 3 mg/kg to 10 mg/kg of the CTLA-4 blocking agent.

In particular embodiments of the combination therapy, the CTLA-4 blocking agent is administered at a dose comprising more than about 10 mg/kg of the CTLA-4 blocking agent.

In particular embodiments of the combination therapy, the PD-1 blocking agent is administered at a dose comprising about 2 or 3 mg/kg or more, or a fixed dose that does not depend on the individual’s weight and is about 200 mg or more.

In particular embodiments of the combination therapy, the PD-1 blocking agent is administered at a dose that does not depend on the individual’s weight that is between 200 mg and 400 mg.

In particular embodiments of the combination therapy, the PD-1 blocking agent is administered at a dose that does not depend on the individual’s weight and is 400 mg.

In particular embodiments of the combination therapy, the PD-1 blocking agent is administered to the individual first and the CTLA-4 blocking agent is administered to the individual second or the CTLA-4 blocking agent is administered to the individual first and the PD-1 blocking agent is administered to the individual second. In a particular embodiment, the Pd-1 blocking agent and the CTLA-4 blocking agent are administered concurrently.

In particular embodiments of the combination therapy, the individual is administered a chemotherapy agent prior to, concurrent with, or subsequent to the combination therapy. In particular embodiments, the chemotherapy agent is selected from the group consisting of actinomycin, all-trans retinoic acid, alitretinoin, azacitidine, azathioprine, bexarotene, bleomycin, bortezomib, carmofur, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabin, hydroxyurea, idarubicin, imatinib, ixabepilone, irinotecan, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, nitrosoureas, oxaliplatin, paclitaxel, pemetrexed, romidepsin, tegafur,

temozolomide(oral dacarbazine), teniposide, tioguanine, topotecan, utidelone, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, and vorinostat.

In particular embodiments of the combination therapy, the cancer is melanoma, non-small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer,

gastrointestinal cancer, multiple myeloma, hepatocellular cancer, non-Hodgkin lymphoma, renal cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, or salivary cancer. In particular embodiments of the combination therapy, the cancer is pancreatic cancer, bronchus cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of hematological tissues.

In particular embodiments of the combination therapy, the individual is a human, the CTLA-4 blocking agent binds a human CTLA-4, the PD-1 blocking agent binds a human PD-1, and the PD-L1 blocking agent binds a human PD-L1.

Antibodies and Compositions

The present invention further provides an effector-silent anti-CTLA-4 antibody or effector-silent anti-CTLA-4 antibody fragment, each comprising a V _j-[ and a VL, wherein the VH comprises three heavy chain CDRs and the VL comprises three light chain CDRs, which together bind CTLA-4. In particular embodiments, the CTLA-4 is a human CTLA-4.

In more specific embodiments, the effector-silent anti-CTLA-4 antibody comprises an IgG _| Fc domain having (i) a mutation in the A-glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes V-glycosylation at said V-glycosylation site or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions with the proviso that the effector-silent anti-CTLA-4 antibody does not include ipilimumab consisting of solely an N297A substitution; (ii) an amino acid substitution mutation selected from the group consisting ofN297A, L234A/L235A/D265A, L234A/L235A/P329G, L235E, D265A, E233A/L235A,S267E/L328F, S2339D/A330L/I332E, L235G/G236R, N297A/D356E/L358M, L234F/L235E/P331S/D365E/L358M, and

D265A/N297G or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; or (iii) a mutation in the N- glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes N- glycosylation at said V-glycosylation site and an amino acid substitution mutation selected from the group consisting of L234A/L235A/D265A, L234A/L235A/P329G, L235E, D265A, E233A/L235A,S267E/L328F, S2339D/A330L/I332E, L235G/G236R, D356E/L358M,

L234F/L235E/P331S/D365E/L358M, and D265A or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

In particular embodiments, the effector-silent anti-CTLA-4 antibody comprises an IgG2 Fc domain having (i) a mutation in the V-glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes V-glycosylation at said V-glycosylation site or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; (ii) an amino acid substitution mutation selected from the group consisting of N297A/D265S, D265A, P329G/D265A/N297G, or

V234A/G237A/P238S/H268A/V309L/A330S/P331S or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; or (iii) a mutation in the /V-glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes /V-glycosylation at said V-glycosylation site and an amino acid substitution mutation selected from the group consisting of N297A/D265S, D265A,

P329G/D265A/N297G, or V234A/G237A/P238S/H268A/V309L/A330S/P331S or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

In particular embodiments, the effector-silent anti-CTLA-4 antibody comprises an IgG4 Fc domain having an S228P amino acid substitution and further comprising (i) a mutation in the /V-glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes /V-glycosylation at said /V-glycosylation site or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; (ii) an amino acid substitution mutation selected from the group consisting of N267A, P329G, and D265A/N297A or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; or (iii) a mutation in the /V- glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes N- glycosylation at said /V-glycosylation site and an amino acid substitution mutation selected from the group consisting of N267A, P329G, and D265A/N297A or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

In particular embodiments, the effector-silent anti-CTLA-4 antibody or effector- silent anti-CTLA-4 antibody fragment comprises the three heavy chain (HC) complementarity determining regions (CDRs) and three light chain (LC) CDRs of an anti-CTLA-4 antibody selected from the group consisting of ipilimumab, tremelimumab, REGN4659, AGEN1884w, 8D2/8D2 (RE), 8D2H1L1, 8D2H2L2, 8D3H3L3, 8D2H2L15, and 8D2H2L17.

In particular embodiments, the effector-silent anti-CTLA-4 antibody or effector- silent anti-CTLA-4 antibody fragment comprises the V _[ and VL of ipilimumab, the V _[ and VL of tremelimumab, the V _[ and VL of REGN4659, the V _[ and V of AGEN1884w, the V _]-[ and V _L of 8D2/8D2 (RE), the V _H and V _L of 8D2H1L1, the V _H and V _L of 8D2H2L2, the V _H and V _L of 8D3H3L3, the V _H and V _L of 8D2H2L15, or the V _H and V _L of 8D2H2L17.

In particular embodiments, the effector-silent anti-CTLA-4 antibody or effector- silent anti-CTLA-4 antibody fragment comprises the V _]-[ and VL of 8D2/8D2 (RE)-Variant 1, the VH and VL of 8D2H1 LI -Variant 1, the V _]-[ and VL of 8D2H2L2 -Variant 1, the V _]-[ and VL of 8D2H2L 15-Variant 1, or the V _]-[ and VL of 8D2H2L17-Variant 1. These variants comprise a substitution of isoleucine for the methionine at position 18 in the V _[ amino acid sequence.

In particular embodiments, the effector-silent anti-CTLA-4 antibody or anti- effector-silent CTLA-4 antibody fragment comprises either (i) a V _j q having the amino acid sequence set forth in SEQ ID NO:7 and a V having the amino acid sequence set forth in SEQ ID NO: 8; (ii) a V _j q having the amino acid sequence set forth in SEQ ID NO: 15 and a VL having the amino acid sequence set forth in SEQ ID NO: 16; (iii) a V _j q having the amino acid sequence set forth in SEQ ID NO:95 and a VL having the amino acid sequence set forth in SEQ ID NO:96; or, (iv) a V _j q having the amino acid sequence set forth in SEQ ID NO:97 and a VL having the amino acid sequence set forth in SEQ ID NO:98.

In particular embodiments, the effector-silent anti-CTLA4 antibody or effector- silent anti-CTLA-4 antibody fragment comprises either (i) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:73 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:74; (ii) a VH domain comprising the amino acid sequence set forth in SEQ ID NO:75 and a VL domain comprising the amino acid sequence set forth in SEQ ID

NO:76; (iii) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:77 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:78; (iv) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:79 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 80; (v) aVp domain comprising the amino acid sequence set forth in SEQ ID NO:81 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 82; (vi) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:83 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 84; (vii) aVp domain comprising the amino acid sequence set forth in SEQ ID NO: 85 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:86; (viii) a Vq domain comprising the amino acid sequence set forth in SEQ ID NO:87 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 88; (ix) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO: 89 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:90; (x) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:91 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:92; or (xi) a Vq domain comprising the amino acid sequence set forth in SEQ ID NO:93 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:94.

In particular embodiments, the effector-silent anti-CTLA-4 antibody fragment is selected from the group consisting of F(ab), F(ab )2- Fv, and scFv.

In a further embodiment, the effector-silent anti-CTLA-4 antibody fragment comprises one or more immunoglobulin single variable domains (ISVDs), each ISVD comprising the variable domain (VHH) °f ^a camelid heavy chain only antibody; with the proviso that none of the ISVDs comprise a VHH that comprises a CDR1 comprising the amino sequence FYGMG (SEQ ID NO:69, a CDR2 comprising the amino acid sequence

DIRTS AGRTTYADSVKG (SEQ ID NO: 70), and a CDR3 comprising amino acid

EMSGISGWDY (SEQ ID NO:71) or EPSGISGWDY (SEQ ID NO:72) as those ISVDs are disclosed in International Patent Application W02008071447, WO2017087587, and

WO2017087588, or a VHH that comprises 1, 2, or 3 mutations in CDR3 as disclosed in

W02008071447, with the exception that ISVDs comprising said CDRs in embodiments wherein the one or more ISVDs are fused or linked to an effector-silent heterologous HC domain or Fc domain, including, for example, any one of the effector-silent antibody HC domains or Fc domains disclosed herein are not excluded by this proviso.

The present invention further provides each of the effector-silent anti-CTLA-4 antibodies disclosed in Tables 4-18 with the proviso that the effector-silent anti-CTLA-4 antibody does not include ipilimumab consisting of solely an N297A substitution.

The present invention further provides a composition comprising an effector- silent anti-CTLA-4 antibody or effector-silent anti-CTLA-4 antibody fragment as disclosed herein and a pharmaceutically acceptable carrier.

The present invention further provides an anti-PD-1 antibody comprising

(a) a heavy chain (HC) having a HC variable domain (V _j q) and a light chain (LC) having a LC variable domain (VL), wherein (i) the V _j q comprises at least the three HC- complementarity determining regions (CDRs) of pembrolizumab and the VL comprises at least the three LC-CDRs of pembrolizumab, (ii) the V _j q comprises at least the three HC-CDRs of nivolumab and the V comprises at least the three LC-CDRs of nivolumab, or (iii) the V _j q comprises at least the three HC-CDRs of cemiplimab-rwlc and the VL comprises at least the three LC-CDRs of cemiplimab-rwlc, and

(b) an IgG _j , IgG2, or IgGq Fc domain comprising (i) a mutation in the N- glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes N- glycosylation at said V-glycosylation site or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; (ii) an IgG _j

Fc domain comprising N297A, L234A/L235A/D265A, L234A/L235A/P329G, L235E, D265A, , E233A/L235A, N297A/D356E/L358M, L234F/L235E/P331S/D356E/L358M, or D265A/N297G amino acid substitutions or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; (iii) an IgG2 Fc domain comprising N297A/D265S, D265A, P329G/D265A/N297G, or

V234A/G237A/P238S/H268A/V309L/A330S/P331S amino acid substitutions or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; or (iv) an IgGq Fc domain comprising an S228P amino acid substitution and an N267A, P329G, D265A/N297A amino acid substitution or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein the amino acid positions are identified according to Eu numbering.

In further still embodiments of the anti-PD-1 antibody above, the anti-PD-1 antibody comprises either (i) a V _j-[ having the amino acid sequence set forth in SEQ ID NO:29 and a VL having the amino acid sequence set forth in SEQ ID NO:30, (ii) a V _j q having the amino acid sequence set forth in SEQ ID NO:23 and a VL having the amino acid sequence set forth in SEQ ID NO:24, or (iii) a V _j q having the amino acid sequence set forth in SEQ ID NO:99 and a V having the amino acid sequence set forth in SEQ ID NO: 100.

In particular embodiments of the anti-PD-1 antibody, the IgG _j , IgG2, or IgGq Fc domain as disclosed herein may further comprise a C-terminal lysine or lack either a C-terminal lysine or a C-terminal glycine-lysine dipeptide.

The present invention further provides an anti-PD-1 antibody fragment comprising a heavy chain (HC) having a HC variable domain (V _j q) and a light chain (LC) having a LC variable domain (VL), wherein (i) the V _j q comprises at least the three HC- complementarity determining regions (CDRs) of pembrolizumab and the VL comprises at least the three LC-CDRs of pembrolizumab, (ii) the V _j q comprises at least the three HC-CDRs of nivolumab and the VL comprises at least the three LC-CDRs of nivolumab, or (iii) the V _j q comprises at least the three HC-CDRs of cemiplimab-rwlc and the VL comprises at least the three LC-CDRs of cemiplimab-rwlc.

In further still embodiments of the anti-PD-1 antibody fragment, the anti-PD-1 antibody fragment comprises either (i) a V _j q having the amino acid sequence set forth in SEQ ID NO:29 and a VL having the amino acid sequence set forth in SEQ ID NO:30, (ii) a V _j q having the amino acid sequence set forth in SEQ ID NO:23 and a VL having the amino acid sequence set forth in SEQ ID NO:24, or (iii) a V _j q having the amino acid sequence set forth in SEQ ID NO: 99 and a VL having the amino acid sequence set forth in SEQ ID NO: 100.

In particular embodiments of the above anti-PD-1 antibody fragments, the anti- PD-1 antibody fragment is selected from the group consisting of F(ab), F(ab )2- Fv, and scFv.

The present invention further provides each of the anti-PD-1 antibodies disclosed in Tables 19 27

The present invention further provides a composition comprising an anti-PD-1 antibody or anti-PD-1 antibody fragment as disclosed herein and a pharmaceutically acceptable carrier.

The present invention further provides an anti-PD-Ll antibody comprising

(a) a heavy chain (HC) having a HC variable domain (V _j q) and a light chain (LC) having a LC variable domain (VL), wherein (i) the V _j q comprises at least the three HC- complementarity determining regions (CDRs) of durvalumab and the VL comprises at least the three LC-CDRs of durvalumab, (ii) the V _j q comprises at least the three HC-CDRs of avelumab and the VL comprises at least the three LC-CDRs of avelumab, or (iii) the V _j-[ comprises at least the three HC-CDRs of atezolizumab and the V comprises at least the three LC-CDRs of atezolizumab, and

(b) an IgG _j , IgG2, or IgG4 Fc domain comprising (i) a mutation in the N- glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes N- glycosylation at said V-glycosylation site or the mutated Fc domain comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; (ii) an IgG _j Fc domain comprising N297A, L234A/L235A/D265A, L234A/L235A/P329G, L235E, D265A, E233A/L235A, N297A/D356E/L358M, L234F/L235E/P331S/D356E/L358M, or D265A/N297G amino acid substitutions or the mutated Fc domain comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; (iii) an IgG2 Fc domain comprising N297A/D265S, D265A, P329G/D265A/N297G, or

V234A/G237A/P238S/H268A/V309L/A330S/P331S amino acid substitutions or the mutated Fc domain comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; or (iv) an IgG4 Fc domain comprising an S228P amino acid substitution and an

N267A, P329G, D265A/N297A amino acid substitution or the mutated Fc domain comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein the amino acid positions are identified according to Eu numbering, with the proviso that when the V _]-[ and VL have the amino acid sequences of SEQ ID NO: 107 and SEQ ID NO: 108, respectively, then the heavy chain (HC) constant domain is not an IgG _j isotype with

N297A/D356E/L358M combination of substitutions or when the V _j q and VL have the amino acid sequences of SEQ ID NO: 103 and SEQ ID NO: 104, respectively, then the HC constant domain is not an IgG _j isotype with L234F/L235E/P331S/D356E/L358M combination of substitutions.

In further still embodiments of the anti-PD-Ll antibody above, the anti-PD-Ll antibody comprises either (i) a V _j q having the amino acid sequence set forth in SEQ ID NO: 103 and a VL having the amino acid sequence set forth in SEQ ID NO: 104, (ii) a V _j q having the amino acid sequence set forth in SEQ ID NO: 105 and a VL having the amino acid sequence set forth in SEQ ID NO: 106, or (iii) a V _j q having the amino acid sequence set forth in SEQ ID NO: 107 and a VL having the amino acid sequence set forth in SEQ ID NO: 108.

In particular embodiments of the anti-PD-Ll antibody, the IgG _j , IgG2, or IgGq

Fc domain as disclosed herein may further comprise a C-terminal lysine or lack either a C- terminal lysine or C-terminal glycine-lysine dipeptide.

The present invention further provides an anti-PD-Ll antibody fragment comprising a heavy chain (HC) having a HC variable domain (V _j q) and a light chain (LC) having a LC variable domain (VL), wherein (i) the V _j-[ comprises at least the three HC- complementarity determining regions (CDRs) of durvalumab and the VL comprises at least the three LC-CDRs of durvalumab, (ii) the V _j-[ comprises at least the three HC-CDRs of avelumab and the V comprises at least the three LC-CDRs of avelumab, or (iii) the V _j-[ comprises at least the three HC-CDRs of atezolizumab and the VL comprises at least the three LC-CDRs of atezolizumab.

In further still embodiments, the anti-PD-Ll antibody fragment comprises either (i) a V _[ having the amino acid sequence set forth in SEQ ID NO: 103 and a VL having the amino acid sequence set forth in SEQ ID NO: 104, (ii) a V _j q having the amino acid sequence set forth in SEQ ID NO: 105 and a VL having the amino acid sequence set forth in SEQ ID NO: 106, or (iii) a V _j q having the amino acid sequence set forth in SEQ ID NO: 107 and a VL having the amino acid sequence set forth in SEQ ID NO: 108.

In particular embodiments of the above anti-PD-Ll antibody fragments, the anti- PD-1 antibody fragment is selected from the group consisting of F(ab), F(ab )2- Fv, and scFv.

The present invention further provides each of the anti-PD-Ll antibodies disclosed in Tables 28-36 with the proviso that when the V _j q and VL have the amino acid sequences of SEQ ID NO: 107 and SEQ ID NO: 108, respectively, then the heavy chain (HC) constant domain is not an IgG _j isotype with N297A/D356E/L358M combination of substitutions or when the V _j q and VL have the amino acid sequences of SEQ ID NO: 103 and SEQ ID

NO: 104, respectively, then the HC constant domain is not an IgG _j isotype with

L234F/L235E/P331S/D356E/L358M combination of substitutions.

The present invention further provides a composition comprising an anti-PD-Ll antibody or anti-PD-Ll antibody fragment disclosed herein and a pharmaceutically acceptable carrier.

The present invention further provides a composition comprising (i) an anti- CTLA-4 antibody disclosed herein and an anti-PD-1 antibody disclosed herein and a

pharmaceutically acceptable carrier; or (ii) an anti-CTLA-4 antibody disclosed herein and an anti-PD-Ll antibody disclosed herein and a pharmaceutically acceptable carrier.

The present invention further provides a composition comprising (i) an anti- CTLA-4 antibody fragment disclosed herein and an anti-PD-1 antibody disclosed herein and a pharmaceutically acceptable carrier or (ii) an anti-CTLA-4 antibody fragment disclosed herein and an anti-PD-Ll antibody disclosed herein and a pharmaceutically acceptable carrier.

The present invention further provides a composition comprising (i) an anti- CTLA-4 antibody fragment disclosed herein and an anti-PD-1 antibody fragment disclosed herein and a pharmaceutically acceptable carrier or (ii) an anti-CTLA-4 antibody fragment disclosed herein and an anti-PD-Ll antibody fragment disclosed herein and a pharmaceutically acceptable carrier. The present invention further provides a composition comprising (i) an anti- CTLA-4 antibody disclosed herein and an anti -PD- 1 antibody fragment disclosed herein and a pharmaceutically acceptable carrier or (ii) an anti-CTLA-4 antibody disclosed herein and an anti- PD-L1 antibody fragment disclosed herein and a pharmaceutically acceptable carrier.

The present invention further provides any one of the anti-CTLA-4, anti-PD-1, or anti-PD-Ll antibodies or compositions as disclosed herein for the treatment of cancer in an individual or for the preparation of a medicament for the treatment of cancer in an individual.

The present invention further provides any one of the anti-CTLA-4, anti-PD-1, or anti-PD-Ll antibody fragments or compositions as disclosed herein for the treatment of cancer in an individual or for the preparation of a medicament for the treatment of cancer in an individual.

In particular embodiments, the cancer is melanoma, non-small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular cancer, non-Hodgkin lymphoma, renal cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, or salivary cancer.

In particular embodiments, the cancer is pancreatic cancer, bronchus cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, adrenal gland cancer, osteosarcoma,

chondrosarcoma, or cancer of hematological tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1A-1E: CTLA-4 blockade mediated colitis is Fc-dependent.

Balb/c mice were treated twice a week with antibodies as indicated for 55 days. Fig. 1A: Gut inflammatory gene expression profiling following administration of an Fc-competent anti- CTLA-4 antibody (a-CTLA4) or effector-silent anti-CTLA-4 antibody have a D265A

substitution (a-CTLA4 (D265S)). After seven weeks of twice weekly treatment, the proximal small intestine was collected for evaluation of gut inflammatory markers by reverse

transcription-quantitative polymerase chain reaction (PCR). A heat map of the fold change in expression of gut inflammatory genes for the two antibodies compared to isotype control treatment is shown. Expression was analyzed in multiple panels and cycle threshold data was normalized to ubiquitin within each panel. Normalized data from genes analyzed as part of multiple panels were averaged prior to determining the fold change over isotype control. Fig.

IB: weight loss over the time of the experiment. Fig. 1C: Intestinal permeability was assessed by measuring FITC-dextran fluorescence in the serum at day 49 and 50. Fig. ID: histologic findings for relative gut inflammation and severity of colitis were examined and scored by a certified pathologist. Fig. IE: representative photomicrographs of hematoxylin and eosin (H & E) stained histological sections of the colon from day 55.

Figs. 2A-2E: Characterization of CTLA-4 ISVD (nAb). Fig. 2A: comparison of the effector-silent CTLA4 ISVD (CTLA4 nAb) to effector-competent a-CTLA4; Fig. 2B:

splenic activated T cells were cultured three days in the presence of CLTA4 nAb or a-CTLA4 as indicated. Proliferation (Fig. 2C), production of IFNy (Fig. 2D), and IL-2 (Fig. 22E) were measured and plotted as fold change over the isotype control (mouse IgG2a)· Data are representative of two-three independent experiment.

Figs. 3A-3D: CTLA4 nAb in combination with an anti-PD-1 antibody (a-PDl) has potent anti-tumoral efficacy. Fig. 3A: CT26 tumor-bearing mice received a dose of the indicated antibody (a-CTLA4, a-PDl, a-CTLA4 (D265A)) at 20 mpk and/or CTLA4 nAb at30 mpk every four days for five doses when tumors reached an average size of 100 mm-’ (ranges 78-125 mm^). Data shows the mean tumor volume over a 32 day period. Results are

representative of two independent experiments (n=10 mice per group); Fig. 3B: CD8 T cells/ Foxp3+ T _re g ratio in the tumor at day one post treatment as indicated. Results are representative of two independent experiments (n=7 mice per group); Fig. 3C and Fig. 3D: Gene expression profile from whole tumor at day eight post treatment. Results are representative of one or two independent experiments (n=5 mice per group). * p<0.05, **p<0.01, *** pO.001 (Unpaired-t test). Error bar ± SEM. Fig. 3E: shows the individual animal tumor volumes for each treatment group compared to isotype controls. Complete responses (CR) through day 39 are presented for responsive treatment groups. Data are representative of two independent experiments with n=10 mice per group.

Figs. 4A-4D: Anti-CTLA-4 antibody mediated colitis is Fc-dependent. Balb/c mice were treated twice a week with antibodies (a-CTLA4, a-PDl, a-CTLA4 (D265A)) alone or in combination with CTLA4 nAb as indicated for 55 days. Fig. 4A: weight loss over the time of the experiment; Fig. 4B: histological assessment of enteritis in proximal jujenum at day 55; Fig. 4C: photomicrographs of H&E stained histological section of the colon; Fig. 4D: shows a heat map of the fold change in expression of gut inflammatory genes for indicated samples compared to isotype control treatment is shown. Expression was analyzed in multiple panels and cycle threshold data was normalized to ubiquitin within each panel. Normalized data from genes analyzed as part of multiple panels was averaged prior to determined fold change over isotype control.

Figs. 5A-5C: Fc effector function Anti-CTLA-4 drives skin but not system inflammation. Balb/c mice were treated twice a week with a-CTLA4 or a-CTLA4 (D265A) as indicated for 55 days. Fig. 5A: Photomicrographs of H&E stained histological section of the ear skin. Fig. 5B: Absolute number of ear skin IL-17-producing T cells, Foxp3+ T _re g cells and neutrophils were measured by flow cytometry. Fig. 5C: photomicrographs of H & E stained histological section of the kidney (top panel), liver (middle panel) and lung (bottom panel). Results are representative of one out two independent experiments (n=4-8 mice per group). Scale bars represent lOOpm. Error bar ± SEM.

Figs. 6A-6D: Fc-sufficient anti-CTLA-4 antibody does not deplete colon Foxp3+ T egs · Fig· 6A: intracellular CTLA-4 staining in CT26-tumor bearing mice in indicated organs.

Fig. 6B: mean Fluorescence Intensity (MFI) of CTLA-4 on Foxp3+ Treg cells. **p<0.01, *** pO.001 (Paired-t test). Fig. 6C and Fig. 6D: Representative dot plot and statistics of colon lamina propria and CT26 tumor infiltrating Foxp3+ Treg 24 hours after treatment as indicated. Data are representative of two to four independent experiments (n=4-12 mice per group) **p<0.01, *** p<0.001 (Unpaired-t test). Error bar ± SEM.

Figs. 7A-7D: Fc-function mediated gut in anti-CTLA-4 impaired T _re g-mediated suppression of colitis. Splenic CD45Rbhigh Naive T cells were transferred into CB17-SCID recipient mice and treated with a-CTLA4 or CTLA4 nAb as indicated. Fig. 7A: weight loss over the time of the experiment. Fig. 7B: photomicrographs of H&E stained histological section of the colon and Fig. 7C: pathology score at day 47(n=14-18 mice per group). Fig. 7D: gene expression profile of the whole colon at day 47 post naive T cell transfer (n=6 mice per group). Data are representative of 1 out 2 independent experiments. Ns=Not Significant **** p<0.0001 (Unpaired-t test). Error bar ± SEM.

Figs. 8A-8E: FcyR engagement and CTLA-4 blockade activate colon macrophages . Fig. 8A and Fig. 8B: CD16/CD32 surface expression on macrophages isolated from spleen, colon lamina propria and tumor from CT26-bearing mice, was assessed by flow cytometry. Fig. 8C: proportion of macrophages (CD45+CDl lb+F4/80+) in the spleen, colon lamina propria and tumor from CT26-bearing mice, was assessed by flow cytometry. Fig. 8D: Illb, Tnfa, I Ghg and Statl mRNA expression was assessed from the colon of mice treated with a- CTLA4, a-CTLA4 (D265A), or CTLA4 nAb at day 0, 10, and 18 post-treatment. Data are representative of two independent experiments (n=8-10 mice per group). ns= not significant, **p<0.01, *** p<0.001 (Paired-t test). Error bar ± SEM. Fig. 8E: Proportion of colon lamina propria IL-17-producing CD4+ T cells (CD45+TCRb+CD4+CD8a-IL-17A+), absolute number of IFNy-producing CD8a+ T cells (C D45+T C Rb+C D4-C D8a+I FNy+) and Neutrophils

(CD45+CD1 lb+Ly6Ghigh) were measured by flow cytometry. Results are representative of 1 out 2 independent experiments (n=4-8 mice per group). **p<0.01 (Unpaired-t test). Scale bars represent 100 pm Error bar ± SEM.

Figs. 9A-9B: Anti-tumor Efficacy in the Mouse Syngeneic MB49 Bladder Tumor Model Study. Fig. 9A: MB49 tumor-bearing mice received a dose of the indicated antibody (30 mg/kg CTLA-4 nAb, 10 mg/kg a-CTLA4, 5 mg/kg a-PDl, or combination of CTLA4 nAb and a-PD-1) every four days for four doses when tumors reached an average size of 102 mnU (ranges 87-117 mm¾. Data shows the mean tumor volume over a 21 day period. Results are representative of two independent experiments (n=10 mice per group). Fig. 9B: shows the individual animal tumor volumes for each treatment group. Complete responses (CR) through day 21 are presented for responsive treatment groups. Data show results from an experiment with n=10 mice per group.

Figs. 10A-10B: Anti-tumor Efficacy in the Mouse Syngeneic MC 38 Colon Tumor Model Study. Fig. 10A: MC38 tumor-bearing mice received a dose of the indicated antibody (30 mg/kg CTLA-4 nAb, 10 mg/kg a-CTLA4, 5 mg/kg a-PDl, or combination of CTLA4 nAb and a-PD-1) every four days for four doses when tumors reached an average size of 220 mrrC (ranges 179-261 mm^). Data shows the mean tumor volume over a 23 day period. Results are representative of two independent experiments (n=10 mice per group). Fig. 10B: shows the individual animal tumor volumes for each treatment group. Complete responses (CR) through day 23 23 are presented for responsive treatment groups. Data show results from an experiment with n=10 mice per group.

Fig. 11: Induction of gut inflammation by effector cells. A cartoon illustrating Fc- mediated induction of gut inflammation can be induced by Effector T cells, independent of T _re g depletion.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

"Adverse event" or“AE” as used herein is set forth in Common Terminology Criteria for Adverse events (CTCAE) Version 5.0, published November 27, 2017, by the U.S. Department of health and Human Services as any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medical treatment with the use of a medical treatment or procedure in a human individual that may or may not be considered related to the medical treatment or procedure. An AE is a term that is a unique representation of a specific event used for medical documentation and scientific analyses. A medical treatment may have one or more associated AEs and each AE may have the same or different level of severity. The severity of an AE is assigned a Grade. The CTCAE displays Grades 1 through 5 with unique clinical descriptions of severity for each AE based on this general guideline: Grade 1, mild, or asymptomatic or mild symptoms, clinical or diagnostic observations only, or intervention not indicated; Grade 2, moderate, or minimal, local or noninvasive intervention indicated, or limiting age-appropriate instrumental activities of daily living (ADL); Grade 3, severe or medically significant but not immediately life-threatening, or hospitalization or prolongation of hospitalization indicated ,or disabling, or limiting self-care (ADL); Grade 4, life-threatening consequences or urgent intervention indicated; and Grade 5, death related to AE. "Antibody" as used herein refers to a glycoprotein comprising either (a) at least two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds, or (b) in the case of a species of camelid antibody, at least two heavy chains (HCs) inter-connected by disulfide bonds. Each HC is comprised of a heavy chain variable region or domain (VH) and a heavy chain constant region or domain. In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region is comprised of three domains, C _]-[ l , C _[ 2 and C p.

In general, the basic antibody structural unit for antibodies is a tetramer comprising two HC/LC pairs, except for the species of camelid antibodies comprising only two HCs, in which case the structural unit is a homodimer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one LC (about 25 kDa) and HC chain (about 50-70 kDa).

In certain naturally occurring antibodies, each light chain is comprised of an LC variable region or domain (VL) and a LC constant domain. The LC constant domain is comprised of one domain, CL. The human VH includes six family members: VH 1 - V _[ 2. V _]-[ 3, VH4, VH5, and VH6; and the human V includes 16 family members: V _K 1, V _K 2, V _K 3, V _K 4, V _K 5, V _K 6, n _l 1, n _l 2, , n _l 3, n _l 4, n _l 5, n _l 6, n _l 7, n _l 8, n _l 9, and n _l 10. Each of these family members can be further divided into particular subtypes. The VH and VL domains can be further subdivided into regions of hypervariability, termed complementarity determining regions

(CDRs), interspersed with regions that are more conserved, termed framework regions (FR).

Each VH and VL is composed of three CDR regions and four FR regions, arranged from amino- terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3,

FR4.

The variable regions of the heavy and light chains contain a binding domain comprising the CDRs that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Rabat, et al. National Institutes of Health, Bethesda, Md. ; 5 ^th ed.; NIH Publ. No. 91-3242 (1991); Rabat (1978) Adv. Prot. Chem. 32: 1-75; Rabat, et al, (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al, (1987)

J Mol. Biol. 196:901-917 or Chothia, et al, (1989) Nature 342:878-883.

Typically, the numbering of the amino acids in the heavy chain constant domain begins with number 118, which is in accordance with the Eu numbering scheme. The Eu numbering scheme is based upon the amino acid sequence of human IgG _j (Eu), which has a constant domain that begins at amino acid position 118 of the amino acid sequence of the IgG _| described in Edelman et al, Proc. Natl. Acad. Sci. USA. 63: 78-85 (1969), and is shown for the IgG _} , IgG2, IgG3, and IgGq constant domains in Beranger, et al., Ed. Ginetoux, Correspondence between the IMGT unique numbering for C-DOMAIN, the IMGT exon numbering, the Eu and Rabat numberings: Human IGHG, Created: 17/05/2001, Version: 08/06/2016, which is accessible at www.imgt.org/IMGTScientificChart/Numbering/ Hu_IGHGnber.html#r).

In general, while a VJ^/VL pair of an antibody comprises six CDRs, three CDRs on the V _j-[ and three CDRs on the VL, the state of the art recognizes that in most cases, the CDR3 region of the heavy chain is the primary determinant of antibody specificity, and examples of specific antibody generation based on CDR3 of the heavy chain alone are known in the art (e.g., Beiboer et al, J. Mol. Biol. 296: 833-849 (2000); Klimka et al, British J. Cancer 83: 252-260 (2000); Rader et al., Proc. Natl. Acad. Sci. USA 95: 8910-8915 (1998); Xu et al, Immunity 13: 37-45 (2000). See Rabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (defining the CDR regions of an antibody by sequence); see also Chothia & Lesk J. Mol. Biol. 196: 901-917 (1987) (defining the CDR regions of an antibody by structure).

The following general rules disclosed in www .bioinf.org.uk : Prof. Andrew C.R. Martin's Group and reproduced in the table below may be used to identify the CDRs in an antibody sequence that comprise those amino acids that specifically interact with the amino acids comprising the epitope in the antigen to which the antibody binds. There are rare examples where these generally constant features do not occur; however, the Cys residues are the most conserved feature.

In general, the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one LC (about 25 kDa) and HC chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy -terminal portion of the HC may define a constant region primarily responsible for effector function of the antibody. Typically, human LCs are classified as kappa and lambda LCs. Furthermore, human HCs are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within LCs and HCs, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the HC also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).

The heavy chain of an antibody may or may not have a terminal lysine (K) residue, or the terminal glycine and lysine (GK) residues. Thus, in particular embodiments of the antibodies herein comprises a heavy chain constant region amino acid sequence shown herein further lacking a terminal lysine and terminating with a glycine residue or further embodiments in which the terminal glycine residue is also lacking. This is because the terminal lysine and sometimes glycine and lysine together may be cleaved during expression of the antibody or cleaved off when introduced into the human body with no apparent adverse effect on antibody efficacy, stability, or immunogenicity. In some cases, the nucleic acid molecule encoding the heavy chain may purposely omit the codons encoding the terminal lysine or the codons for the terminal lysine and glycine.

"Antibody fragment” or“Antigen binding fragment" as used herein refers to fragments of full-length antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody but are less than full-length and which either lack an Fc domain in its entirety or lack those portions of the Fc domain that confer binding of the antibody to the FcyRs. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; scFv molecules; NANOBODIES, and multispecific antibodies formed from antibody fragments.

"Chimeric antibody" as used herein is an antibody having the variable domain from a first antibody and the constant domain from a second antibody wherein (i) the first and second antibodies are from different species (U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)) or (ii) the first and second antibodies are from different isotypes, e.g., variable domain from an IgG _| antibody and the constant domains from an IgGq antibody). In one aspect, the variable domains are obtained from a non-human antibody such as a mouse antibody (the "parental antibody"), and the constant domain sequences are obtained from a human antibody. In a further aspect, the variable domains are humanized variable domains from a mouse antibody and the constant domains of a human antibody.

“Combination therapy” as used herein refers to treatment of a human or animal individual comprising administering a first therapeutic agent and a second therapeutic agent consecutively or concurrently to the individual. In general, the first and second therapeutic agents are administered to the individual separately and not as a mixture; however, there may be embodiments where the first and second therapeutic agents are mixed prior to administration.

"Conservative substitution" as used herein refers to substitutions of amino acids with other amino acids having similar characteristics (e.g. charge, side-chain size,

hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that the changes can frequently be made without altering the biological activity of the protein. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.) (1987)). In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are set forth in Table 2.

"Cytotoxic T lymphocyte-associated antigen-4," "CTLA-4," "CTLA4," "CTLA-4 antigen" and "CD152" (see, e.g., Murata, Am. J. Pathol. 155:453-460 (1999)) are used interchangeably, and include variants, isoforms, species homologs of human CTLA-4, and analogs having at least one common epitope with CTLA-4 (see, e.g., Balzano, Int. J. Cancer Suppl. 7:28-32 (1992)). The complete CTLA-4 nucleic acid sequence can be found under GenBank Accession No. L15006.

"Effector function" as used herein refers to those biological activities attributable to the Fc region of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC);

phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation. Antibodies act by a number of mechanisms, most of which engage other arms of the immune system. Antibodies can simply block interactions of molecules or they can activate the classical complement pathway (known as complement dependent cytotoxicity or CDC) by interaction of Clq on the Cl complex with clustered antibodies. Critically antibodies also act as a link between the antibody-mediated and cell-mediated immune responses through engagement of Fc receptors. “Effector-silent” as used herein refers to an antibody or antibody fragment that displays (i) no measurable binding to one or more Fc receptors (FcRs) as may be measured in a Biacore assay wherein an association constant in the micromolar range indicates no measurable binding or (ii) measurable binding to one or more FcRs as may be measured in a Biacore assay that is reduced compared to the binding that is typical for an antibody of the same isotype. In particular embodiments, the antibody may comprise one or more mutations in the HC constant domain and the Fc domain in particular such that the mutated antibody has reduced or no measurable binding to FcyRIIIa, FcyRIIa, and FcyRI compared to a wild-type antibody of the same isotype as the mutated antibody. In particular embodiments, the affinity or association constant of an effector-silent antibody to one or more of FcyRIIIa, FcyRIIa, and FcyRI is reduced by at least 1000-fold compared to the affinity of the wild-type isotype; reduced by at least 100- fold to 1000-fold compared to the affinity of the wild-type isotype reduced by at least 50-fold to 100-fold compared to the affinity of the wild-type isotype; or at least 10-fold to 50-fold compared to the affinity of the wild-type isotype. In particular embodiments, the effector-silent antibody has no detectable or measurable binding to one or more of the FcyRIIIa, FcyRIIa, and FcyRI as compared to binding by the wild-type isotype. In general, effector-silent antibodies will lack measurable antibody-dependent cell-mediated cytotoxicity (ADCC) activity. An effector- silent antibody fragment lacks an Fc domain or those portions of an Fc domain that confer binding to FcRs and as such would display no detectable or measurable binding to one or more of FcyRIIIa, FcyRIIa, or FcyRI. For effector-silent antibody or antibody fragments, the binding is measured against human FcRs.

"Fab fragment" as used herein comprises of one LC and the V _j-[ and C _[ 1 of one HC and excludes the remainder of the HC constant domain. The CHI of the Fab molecule cannot form a disulfide bond with another Fab fragment or HC containing molecule. A "Fab fragment" can be the product of papain cleavage of an antibody.

"Fab' fragment" as used herein comprises one LC and a fragment of one HC that contains the V _j-[ domain and the HC constant domain up to a region between the C _[ 1 and C _[ 2 domains and excludes the remainder of the HC constant domain, such that an inter-chain disulfide bond can be formed between the two HCs of two Fab' fragments to form a F(ab')2 molecule.

"F(ab')2 fragment" as used herein comprises two LCs and two HC fragments, each HC fragment containing the V _j-[ domain and the HC constant domain up to a region between the C _j ^l and C _[ 2 domains and excludes the remainder of the HC constant domain, such that an inter-chain disulfide bond is formed between the two HCs. A F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains. An F(ab')2 fragment may be obtained by digesting an antibody with pepsin, which cleaves the antibody at a site between the Cpfland C 2 domains. "Fc domain”, or“Fc” as used herein is the crystallizable fragment domain or region obtained from an antibody that comprises the C\ l and C p domains of an antibody. In an antibody, the two Fc domains are held together by two or more disulfide bonds and by hydrophobic interactions of the C p domains. The Fc domain may be obtained by digesting an antibody with the protease papain.

“Fc receptors” or“FcRs” as used herein are key immune regulatory receptors connecting the antibody mediated (humoral) immune response to cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcyR (IgG), FcsRI (IgE), FcaRI (IgA), FcpR (IgM) and Fc5R (IgD). There are three classes of receptors for human IgG found on leukocytes: CD64 (FcyRI), CD32 (FcyRIIa. FcyRIIb and FcyRIIc) and CD 16 (FcyRIIIa and FcyRIIIb). FcyRI is classed as a high affinity receptor (nanomolar range KD) while FcyRI I and FcyRIII are low to intermediate affinity (micromolar range KD). In antibody dependent cellular cytotoxicity (ADCC), FcRs on the surface of effector cells (natural killer cells, macrophages, monocytes and eosinophils) bind to the Fc region of an IgG which itself is bound to a target cell. Upon binding a signaling pathway is triggered which results in the secretion of various substances, such as lytic enzymes, perforin, granzymes and tumor necrosis factor, which mediate in the destruction of the target cell. The level of ADCC effector function various for human IgG subtypes. Although this is dependent on the allotype and specific FcR in simple terms ADCC effector function is high for human IgG _[ and IgG3, and low for IgG2 and

IgGzp

"Fv region" as used herein comprises a single VH and VL pair wherein the VH polypeptide and the VL polypeptide are held together by disulfide bonds.

"Humanization" (also called Reshaping or CDR-grafting) as used herein is a well-established technique for reducing the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (commonly rodent) and for improving the effector functions (ADCC, complement activation, Clq binding). The engineered mAh is engineered using the techniques of molecular biology, however simple CDR-grafting of the rodent complementarity-determining regions (CDRs) into human frameworks often results in loss of binding affinity and/or specificity of the original mAh. In order to humanize an antibody, the design of the humanized antibody includes variations such as conservative amino acid substitutions in residues of the CDRs, and back substitution of residues from the rodent mAh into the human framework regions (back mutations). The positions can be discerned or identified by sequence comparison for structural analysis or by analysis of a homology model of the variable regions' three-dimensional structure. The process of affinity maturation has most recently used phage libraries to vary the amino acids at chosen positions. Similarly, many approaches have been used to choose the most appropriate human frameworks in which to graft the rodent CDRs. As the datasets of known parameters for antibody structures increases, so does the sophistication and refinement of these techniques. Consensus or germline sequences from a single antibody or fragments of the framework sequences within each light or heavy chain variable region from several different human mAbs can be used. Another approach to humanization is to modify only surface residues of the rodent sequence with the most common residues found in human mAbs and has been termed

"resurfacing" or "veneering." Often, the human or humanized antibody is substantially non- immunogenic in humans.

"Humanized antibody" as used herein refers to forms of antibodies or antibody fragments that contain sequences from both human and non-human (e.g., murine, rat) antibodies. In general, the humanized antibody will comprise all of at least one, and typically two, variable domains, in which the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (e.g., Fc domain).

"Hyperproliferative disease" as used herein refers to conditions wherein cell growth is increased over normal levels. For example, hyperproliferative diseases or disorders include malignant diseases (e.g., esophageal cancer, colon cancer, biliary cancer) and non- malignant diseases (e.g., atherosclerosis, benign hyperplasia, benign prostatic hypertrophy).

“Immune-related adverse events” or irAE” as used herein refers to AEs that are autoimmune manifestations due to unbalancing the immune system as may be attributed to use of one or more immune checkpoint inhibitors such as anti-PD-1, anti-PD-Ll, and anti-CTLA-4 antibodies.

"Immune response" as used herein refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble

macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that result in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

“Immunoglobulin single variable domain” (also referred to as“ISV” or

ISVD”) as used herein is generally used to refer to immunoglobulin variable domains (which may be heavy chain or light chain domains, including Vpp Vp _[ H ^or Vp domains) that can form a functional antigen binding site without interaction with another variable domain (e.g. without a VR/VE interaction as is required between the V _j q and Vp domains of conventional 4-chain monoclonal antibody). Examples of ISVDs include NANOBODIES (including a Vp _[ H _> a humanized VHH and/or a camelized Vp _[S such as camelized human Vp _[S ). IgNAR, domains, (single domain) antibodies (such as dAbs™) that are Vp _[ domains or that are derived from a Vp _[ domain and (single domain) antibodies (such as dAbs™) that are Vp domains or that are derived from a VL domain. ISVDs that are based on and/or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred.

"Monoclonal antibody" as used herein refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains that are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al, Nature 256: 495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature 352: 624-628 (1991) and Marks et al, J. Mol. Biol. 222: 581-597 (1991), for example. See also Presta J. Allergy Clin. Immunol. 116:731 (2005).

“NANOBODY” and“NANOBODIES” as used herein are registered trademarks of Ablynx N.V.

“Non-human amino acid sequences” as used herein with respect to antibodies or immunoglobulins refers to an amino acid sequence that is characteristic of the amino acid sequence of a non-human mammal. The term does not include amino acid sequences of antibodies or immunoglobulins obtained from a fully human antibody library where diversity in the library is generated in silico (See for example, U.S. Pat. No. 8,877,688 or 8,691,730).

“PD-1” refers to the programmed Death 1 ( PD-1 ) protein, an inhibitory member of the extended CD28/CTLA-4 family of T cell regulators (Okazaki et al, Curr. Opin. Immunol. 14: 391779-82 (2002); Bennett et al, J. Immunol. 170:711-8 (2003)). Other members of the CD28 family include CD28, CTLA-4, ICOS and BTLA. The PD-1 gene encodes a 55 kDa type I transmembrane protein (Agata et al, Inti. Immunol. 8:765-72 (1996)). Two ligands for PD-1 have been identified, PD-L1 (B7-H1) and PD-L2 (B7-DC), that have been shown to

downregulate T cell activation upon binding to PD-1 (Freeman et al. (2000) J. Exp. Med.

192: 1027-34; Carter et al. (2002) Eur. J. Immunol. 32:634-43). PD-1 is known as an immunoinhibitory protein that negatively regulates TCR signals (Ishida, Y. et al, EMBO J. 11:3887-3895 (1992); Blank, C. et al, Immunol. Immunother. 56(5):739-745 (Epub 2006 Dec. 29)). The interaction between PD-1 and PD-L1 can act as an immune checkpoint, which can lead to, e.g., a decrease in tumor infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and/or immune evasion by cancerous cells (Dong et al, J. Mol. Med. 81:281-7 (2003); Blank et al, Cancer Immunol. Immunother. 54:307-314 (2005); Konishi et al, Clin. Cancer Res. 10:5094-100 (2004)). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2; the effect is additive when the interaction of PD-1 with PD-L2 is blocked as well (Iwai et al, Proc. Nat'l. Acad. Sci. USA 99: 12293-12297 (2002); Brown et al, J. Immunol. 170: 1257-66 (2003)).

"Programmed Death 1," "Programmed Cell Death 1," "Protein PD-1," "PD-1" "PD1," "PDCDl,""hPD-l" and "hPD-1" are used interchangeably, and include variants, isoforms, species homologs of human PD-1, and analogs having at least one common epitope with PD-1. The complete PD-1 sequence can be found under GenBank Accession No. U64863.

“ScFv” or“single-chain variable fragment” as used herein is a fusion protein comprising a V _j q and VL fused or linked together by a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the V-terminus of the V _j q with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.

"Subtherapeutic dose" as used herein means a dose of a therapeutic compound (e.g., an antibody) that is lower than the usual or typical dose of the therapeutic compound when administered alone for the treatment of a hyperproliferative disease (e.g., cancer). The dose of a therapeutic compound may vary depending on the disease being targeted. For example, a subtherapeutic dose of CTLA-4 antibody is a single dose of the antibody at less than about 3 mg/kg, i.e., the known monotherapy dose of anti-CTLA-4 antibody YERVOY for treatment of unresectable or metastatic melanoma, or a single dose of YERVOY at less than about 10 mg/kg, the known monotherapy dose for adjuvant melanoma.

"Treat" or "treating" as used herein means to administer a therapeutic agent, such as a composition containing any of the antibodies or antigen binding fragments thereof of the present invention, internally or externally to a subject or patient having one or more disease symptoms, or being suspected of having a disease, for which the agent has therapeutic activity or prophylactic activity. Typically, the agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing the regression of or inhibiting the progression of such symptom(s) by any clinically measurable degree. The amount of a therapeutic agent that is effective to alleviate any particular disease symptom may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired response in the subject. Whether a disease symptom has been alleviated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom. The term further includes a postponement of development of the symptoms associated with a disorder and/or a reduction in the severity of the symptoms of such disorder. The terms further include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result has been conferred on a human or animal subject with a disorder, disease or symptom, or with the potential to develop such a disorder, disease or symptom.

“Therapeutically effective amount” as used herein refers to a quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this may be the amount of CTLA-4 blocking agent necessary to inhibit activation of CTLA-4 and induce an anti-tumor response or the amount necessary for enhanced anti-PD-1 or PD-L1 responsiveness when co-administered with anti-PD-1 or anti-PD-Ll blocking agent, respectively.

“Therapeutic index”, also known as“therapeutic window”,“safety window” or“therapeutic ratio” as used herein is a comparison of the amount of a therapeutic agent that causes a therapeutic effect to the amount of the therapeutic agent that causes toxicity.

"Treatment" as it applies to a human or veterinary individual, as used herein refers to therapeutic treatment, which encompasses contact of antibodies or antigen binding fragments to a human or animal individual who is in need of treatment with the antibodies or antibody fragments.

VHH ^{as use} d herein indicates that the V _j q domain is obtained from or originated or derived from a HC antibody. Heavy chain antibodies are functional antibodies that have two HCs and no LCs. Heavy chain antibodies exist in and are obtainable from Camelids, members of the biological family Camelidae.

Introduction

PD-1 antagonists such as the commercially marketed anti-PD-1 antibodies KEYTRUDA and OPDIVO comprise a human IgGq backbone, which has reduced FcyR function, because pre-clinical studies with anti-PD-1 antibodies with FcyR binding function showed poor anti-tumor efficacy due to depletion of CD8+ cytotoxic T cells (CTL), which are essential for tumor immunotherapy (See e.g., International Patent Application W02014/089113). In contrast, monotherapies using anti-CTLA-4 antibodies were shown in pre-clinical experiments that compared mouse IgG2 _a -anti-CTLA-4 antibodies, which have high FcyR-binding affinity, with mutant mouse IgG _j -anti-CTLA-4 antibodies, which lack measurable FcyR-binding affinity, to require FcyR function in order to effect strong anti-tumor y responses (See e.g., Selby et al, Cancer Immunol Res. 1:32-42 (2013). The requirement for FcyR function in the anti-CTLA-4 antagonist monotherapy correlated with depletion of T regulatory cells (T _re g) in murine tumor models due to higher CTLA-4 expression on TILs (Simpson et al, J. Exp. Med. 210:1695-710 (2013)) compared to T _re g populations in spleen or lymph nodes.

The inventors of the instant invention hypothesized that the requirement for FcyR function for anti-CTLA-4 antibody efficacy may be circumvented by combining the anti-CTLA- 4 antibody with an anti-PD-1 antibody. This hypothesis is supported by emerging data illustrating a critical role for CD28-mediated co-stimulation in anti-PD-1 -mediated activation of exhausted CD8 ⁺ cytotoxic T cells. Anti-CTLA-4 and anti-PD-1 antibodies exert their anti-tumor activities via different mechanisms. Importantly, the combination effects of the anti-CTLA-4 and anti-PD-1 antibodies are not merely additive, as the combined blockade exerted by the two antibodies results in activation of a large number of genes, including proliferation-associated and chemokine genes, that are not activated by either antibody alone (see for Example Figs. 3C and 4D). These data suggest that the mechanism of action for the CTLA-4 blockade in a

monotherapy differs from its mechanism of action when performed in combination with the PD- 1 blockade.

Emerging data indicates the importance of CD28-mediated co-stimulation in activating effector T (T _e ff) cells following the PD-1 blockade. PD-1 signaling dephosphorylates

CD28, rather than TCR as previously assumed, and CD28 signaling is required for the enhanced anti-tumor response observed following the PD-1 blockade. Therefore, while a monotherapy CTLA-4 blockade may primarily target T cell priming events, combining the CTLA-4 blockade with a PD-1 blockade can be expected to facilitate activation of exhausted T cells beyond what would be expected from a PD-1 blockade alone. The inventors postulated that this mechanism is enhanced by CTLA-4 antagonists, which enables increased CD28-mediated activation, independent of the function of Fc-receptors, and the depletion of T _re g cells, which may play an important factor for irAE mediated toxicity.

A potential caveat then for anti-CTLA-4 antibodies that bind FcRs (Fc-functional antibodies) is that T _re g depletion or cell bridging of myeloid cells with T cells may induce undesired immune-related inflammation. The inventors hypothesized that it is Fc function that may be contributing to the observed irAEs associated with CTLA-4 blockade cancer

immunotherapy. One critique that has been used to argue against the potential role of Fc function for the induction of irAEs has been that both ipilimumab (on a human IgG _| backbone) and tremelimumab (on a human IgG2 backbone) treatment are associated with gut inflammation. While the human IgG2 Fc domain has significantly lower affinity for human FcyRs compared to human IgG _j , direct comparison of antibodies with human IgG2 and IgG _| backbones have shown that both elicit similar levels of Fc function using in vitro ADCC and ADCP bioassays (e.g., Vargas et al, Cancer Cell. 33: 649-663 (2018)). Moreover, in vivo T _re g depletion and anti tumor activity of chimeric anti-mouse CTLA-4 antibodies with either a human IgG _j isotype backbone or a human IgG2 isotype backbone were equivalent in human FcyR knock-in mice

(Vargas et al, ibid.).

A key impediment for assessing the potential role of Fc function for inducing gut inflammation in syngeneic tumor models has been the lack of measurable inflammation and colitis using mouse anti-CTLA-4 surrogate antibodies. To circumvent this impediment, the inventors have employed a PCR-based panel that was previously developed by Cayatte et al, Clin. Transl. Gastroenterol. 3: elO (2012) to measure upregulation of gut inflammatory genes associated with inflammatory bowel disease (IBD) in a mouse IBD model. As shown in the examples herein, this PCR-based panel enabled the inventors to detect increased expression of biomarker genes indicative of gut inflammation in mice treated with an Fc functional anti-mouse CTLA-4 antibody (a-CTLA4), even in the absence of overt colitis or histological evidence of tissue damage (See Figs. 1A, 3C, and 4D. This observation of subclinical stimulation of gut inflammation gene expression pathways inspired the inventors to extend the treatment schedule to determine if the underlying inflammation would progress to development of clinical colitis. This irAE colitis mouse model enabled the inventors to run empirical experiments to assess the requirement of Fc function for induction of gut inflammation in the context or absence of concomitant anti-tumor responses in syngeneic tumor models utilized in immuno-oncology preclinical development.

The results described in the examples clearly show that in a monotherapy setting, neither an effector-silent anti-CTLA-4 antibody nor an effector-silent anti-CTLA-4 antibody fragment elicits measurable anti -tumor activity. However, administering the effector-silent antibody or effector-silent antibody fragment in combination with an anti-PDl antibody results in antitumor activity that is comparable to the anti-tumor activity elicited by an effector-functional anti-CTLA-4 antibody either alone or in combination with an anti-PD-1 antibody (See Fig. 3A) and without the gut or skin irAEs observed for the effector-functional anti-CTLA-4 antibody alone or in combination with an anti-PD-1 antibody (Fig. 4B and 5A) or loss of weight (Fig.

4A). In light of these results and the inventors’ discovery of a potential T _re g-independent mechanism associated with Fc-mediated anti-tumor activity and gut-inflammation, the present invention makes possible CTLA-4/ PD-1 blockade combination anti-cancer immunotherapies with improved therapeutic index and broader utility.

Combination Therapies

The present invention provides anti-cancer combination therapies, which comprise, administering to an individual in need of a cancer therapy (i) a PD-1 blocking agent selected from the group consisting of an anti-PD-1 antibody, an anti-PD-Ll antibody, an effector-silent anti-PD-1 antibody, an effector-silent anti-PD-Ll antibody, an effector-silent anti- PD-1 antibody fragment, and an effector-silent anti-PD-Ll antibody fragment; and, (ii) an effector-silent CTLA-4 blocking agent selected from the group consisting of an effector-silent anti-CTLA-4 antibody and an effector-silent anti-CTLA-4 antibody fragment.

The effector-silent CTLA-4 blocking agent may be administered in a combination therapy with a PD-1 blocking agent at doses that are greater than the subtherapeutic 1 mg/kg dose of ipilimumab approved by the U.S. FDA for ipilimumab/nivolumab combination therapies targeting advance renal cell carcinoma or microsatellite instability -high or mismatch repair deficient metastatic colorectal cancer and with a lower risk of inducing skin or gut irAEs greater than Grade 1-2 according to the criteria set forth in Common Terminology Criteria for Adverse events (CTCAE) Version 5.0, for the duration of the combination therapy or for at least a portion of the time period the individual is undergoing the combination therapy than is observed for the ipilimumab/nivolumab combination therapies. In particular embodiments, the doses do not induce irAEs greater than Grade 1 for the duration of the combination therapy or for at least a portion of the time period the individual is undergoing the combination therapy.

Thus, in particular embodiments, the effector-silent CTLA-4 blocking agent may be administered to an individual at a dose greater than 1 mg/kg. In particular embodiments, the effector-silent CTLA-4 blocking agent may be administered to an individual at a dose of at least 3 mg/kg. In particular embodiments, the effector-silent CTLA-4 blocking agent may be administered to an individual at a dose of at least 10 mg/kg. In particular embodiments, the effector-silent CTLA-4 blocking agent may be administered to an individual at a dose of at least 15 mg/kg. In particular embodiments, the effector-silent CTLA-4 blocking agent may be administered to an individual at a dose of at least 20 mg/kg. In particular embodiments, the effector-silent CTLA-4 blocking agent may be administered to an individual at a dose between 3 mg/kg and 20 mg/kg. In particular embodiments, the effector-silent CTLA-4 blocking agent may be administered to an individual at a fixed dose that does not depend on the individual’s weight, for example, a dose that is greater than 100 mg.

In particular embodiments of the combination therapy, the effector-silent CTLA-4 blocking agent is an effector-silent anti-CTLA-4 antibody or (b) effector-silent anti-CTLA-4 antibody fragment. Because effector function activity is not wanted for the anti-CTLA-4 antibody, the anti-CTLA-4 antibody either has an HC domain that has been engineered to be “effector-silent”, that is, modifying its Fc domain to have reduced or no measurable FcR binding compared to the Fc domain of a wild-type antibody of the same isotype as the effector-silent antibody (e.g., Fc domain of non-mutated IgG _| . IgG2, IgG3, or IgG4 Fc domain) as determined by a Biacore assay. An effector-silent anti-CTLA-4 antibody fragment either lacks an Fc domain or those regions of the Fc domain that bind one or more FcRs.

In particular embodiments, the combination therapy of the present invention is administered to an individual prior to or subsequent to surgery to remove a tumor and may be used before, during, or after radiation therapy.

In particular embodiments, the combination therapy of the present invention is administered to an individual who has not been previously treated with a biotherapeutic or chemotherapeutic agent, i.e., the individual is treatment-naive. In other embodiments, the combination therapy is administered to an individual who has failed to achieve a sustained response after a prior therapy with a biotherapeutic or chemotherapeutic agent, i.e., the individual is treatment-experienced.

In particular embodiments, the combination therapy of the present invention is used to treat a tumor that is large enough to be found by palpation or by imaging techniques well known in the art, such as MRI, ultrasound, or CAT scan. In some embodiments, a combination therapy of the invention is used to treat an advanced stage tumor having dimensions of at least about 200 mm^· 300 mm^, 400 mm^, 500 mm^, 750 mm^, or up to 1000 mm^.

In particular embodiments, the combination therapy of the present invention is administered to an individual who has a cancer that tests positive for PD-L1 expression. In some embodiments, PD-L1 expression is detected using a diagnostic anti -human PD-L1 antibody, or antigen binding fragment thereof, in an immunohistochemical (IHC) assay on fixed formalin paraffin embedded (FFPE) or frozen tissue section of a tumor sample removed from the individual. An individual’s physician may order a diagnostic test to determine PD-L1 expression in a tumor tissue sample removed from the individual prior to initiation of treatment with combination therapy of the present invention but it is envisioned that the physician could order the first or subsequent diagnostic tests at any time after initiation of treatment, such as for example, after completion of a treatment cycle.

The combination therapy may comprise any one of the exemplary effector-silent anti-CTLA-4 antibodies or effector-silent anti-CTLA-4 antibody fragments disclosed herein in combination with any one of the exemplary anti-PD-1 antibodies or anti-PD-1 antibody fragments disclosed herein or any one of the exemplary anti-PD-Ll antibodies or anti-PD-Ll antibody fragments disclosed herein.

(a) Effector -silent antibodies

An effector-silent antibody of the present invention comprises an HC constant domain or Fc domain thereof that has been modified such that the antibody displays no measurable binding to one or more FcRs or displays reduced binding to one or more FcRs compared to that of an unmodified antibody of the same IgG isotype. The effector-silent antibodies may in further embodiments display no measurable binding to each of FcyRIIIa, FcyRIIa. and FcyRI or display reduced binding to each of FcyRIIIa, FcyRIIa. and FcyRI compared to that of an unmodified antibody of the same IgG isotype. In particular embodiments, the HC constant domain or Fc domain is a human HC constant domain or Fc domain.

In particular embodiments, the effector-silent antibody comprises an Fc domain of an IgG _} or IgG2, IgG3, or IgGq isotype that has been modified to lack A-glycosylation of the asparagine (Asn) residue at position 297 (Eu numbering system) of the HC constant domain.

The consensus sequence for A-glycosylation is Asn-Xaa-Ser/Thr (wherein Xaa at position 298 is any amino acid except Pro); in all four isotypes the X-glycosylation consensus sequence is Asn- Ser-Thr. The modification may be achieved by replacing the codon encoding the Asn at position 297 in the nucleic acid molecule encoding the HC constant domain with a codon encoding another amino acid, for example Ala, Asp, Gin, Gly, or Glu, e.g. N297A, N297Q, N297G, N297E, or N297D. Alternatively, the codon for Ser at position 298 may be replaced with the codon for Pro or the codon for Thr at position 299 may be replaced with any codon except the codon for Ser. In a further alternative each of the amino acids comprising the A-glycosylation consensus sequence is replaced with another amino acid. Such modified IgG molecules have no measurable effector function. In particular embodiments, these mutated HC molecules may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations. In further embodiments, such IgGs modified to lack/V-glycosylation at position 297 may further include one or more additional mutations disclosed herein for eliminating measurable effector function.

An exemplary IgG _j HC constant domain mutated at position 297, which abolishes the A-glycosylation of the HC constant domain, is set forth in SEQ ID NO:44, an exemplary IgG2 HC constant domain mutated at position 297, which abolishes the N- glycosylation of the HC constant, is set forth in SEQ ID NO:50, and an exemplary IgGq HC constant domain mutated at position 297 to abolish /V-glycosylation of the HC constant domain is set forth in SEQ ID NO:56. In particular embodiments, these mutated HC molecules may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In particular embodiments, the Fc domain of the IgG _j IgG2, IgG3, or IgGq HC constant domain comprising the effector-silent antibody is modified to include one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S (wherein the positions are identified according to Eu numbering) and wherein said HC constant domain is effector-silent. In particular embodiments, the modified IgG _| further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In particular embodiments, the HC constant domain comprises L234A, L235A, and D265S substitutions (wherein the positions are identified according to Eu numbering). In particular embodiments, the HC constant domain comprises an amino acid substitution at position Pro329 and at least one further amino acid substitution selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S (wherein the positions are identified according to Eu numbering). These and other substitutions are disclosed in WO9428027; W02004099249; W020121300831, U.S. Pat. Nos. 9,708,406; 8,969,526; 9,296,815;

Sondermann et al. Nature 406, 267-273 (20 Jul. 2000)).

In particular embodiments of the above, the HC constant domain comprises an L234A/L235A/D265A; L234A/L235A/P329G; L235E; D265A; D265A/N297G; or

V234A/G237A/P238S/H268A/V309L/A330S/P331S substitutions, wherein the positions are identified according to Eu numbering. In particular embodiments, the HC molecules further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In particular embodiments, the effector-silent antibody comprises an IgG _j isotype, in which the Fc domain of the HC constant domain has been modified to be effector- silent by substituting the amino acids from position 233 to position 236 of the IgG _] with the corresponding amino acids of the human IgG2 HC and substituting the amino acids at positions 327, 330, and 331 with the corresponding amino acids of the human IgG4 HC, wherein the positions are identified according to Eu numbering (Armour et al, Eur. J. Immunol. 29(8):2613- 24 (1999); Shields et al, J. Biol. Chem. 276(9):6591-604(2001)). In particular embodiments, the modified IgG _| further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In particular embodiments, the effector-silent antibody comprises a V _[ domain fused or linked to a hybrid human immunoglobulin HC constant domain, which includes a hinge region, a C¾ domain and a CH3 domain in an V-terminal to C-terminal direction, wherein the hinge region comprises an at least partial amino acid sequence of a human IgD hinge region or a human IgG _j hinge region; and the C¾ domain is of a human IgGq C¾ domain, a portion of which, at its V-terminal region, is replaced by 4-37 amino acid residues of an V-terminal region of a human IgG2 C¾ or human IgD C¾ domain. Such hybrid human HC constant domain is disclosed in U.S. Pat. No. 7,867,491, which is incorporated herein by reference in its entirety.

In particular embodiments, the effector-silent antibody comprises an IgGq HC constant domain in which the serine at position 228 according to the Eu system is substituted with proline, see for example SEQ ID NO: 52. This modification prevents formation of a potential inter-chain disulfide bond between the cysteines at positions Cys226 and Cys229 in the EU system and which may interfere with proper intra-chain disulfide bond formation. See Angal et al. Mol. Imunol. 30: 105 (1993); see also (Schuurman et al, Mol. Immunol. 38: 1-8, (2001); SEQ ID NOs: 14 and 41). In further embodiments, the IgGq constant domain includes in addition to the S228P substitution, a P239G, D265A, or D265A/N297G amino acid substitution, wherein the positions are identified according to Eu numbering. In particular embodiments of the above, the IgGq HC constant domain is a human HC constant domain. In particular embodiments, the HC molecules further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

Exemplary IgG _] HC constant domains include HC constant domains comprising an amino acid sequence selected from the group consisting of amino acid sequences set forth in SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44. Exemplary IgG2 HC constant domains have an amino acid sequence selected from the group consisting of amino acid sequences set forth in SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49. Exemplary IgG ₄ HC constant domains have an amino acid sequence selected from the group consisting of amino acid sequences set forth in SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56.

More specific examples of effector-silent antibodies are described below in combination with particular exemplary effector-silent anti-CTLA-4 antibodies, anti-PD-1 antibodies, and anti-PD-1 antibodies.

(b) Exemplary effector -silent anti-CTLA-4 antibodies

Exemplary effector-silent anti-CTLA-4 antibodies that may be used in the combination therapy of the present invention and compositions comprising these antibodies include any effector-silent anti-CTLA-4 antibody that binds CTLA-4 and inhibits CTLA-4 from binding B7. Specific effector-silent anti-CTLA-4 antibodies include the following effector silent anti-CTLA-4 antibodies and compositions comprising any one of these antibodies and a pharmaceutically acceptable carrier.

In particular embodiments, the effector-silent anti-CTLA-4 antibody comprises (i) a V _j q comprising the three HC-CDRs of ipilimumab fused or linked to an HC constant domain that displays no measurable binding to the FcyRIIIA, FcyRIIA, and FcyRI or reduced binding compared to a polypeptide comprising the wild-type IgG constant domain region as determined by a Biacore assay and (ii) a VL comprising the three LC-CDRs of ipilimumab fused or linked to an LC kappa or lambda constant domain. The three HC-CDRs comprise SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, and the three LC-CDRs comprise SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3, respectively.

In further embodiments, the effector-silent anti-CTLA-4 antibody comprises (i) a V _j q comprising the three HC-CDRs of tremelimumab fused or linked to an HC constant domain that displays no measurable binding to the FcyRIIIA, FcyRIIA, and FcyRI or reduced binding compared to a polypeptide comprising the wild-type IgG constant domain region as determined by a Biacore assay and (ii) a VL comprising the three LC-CDRs of tremelimumab fused or linked to an LC kappa or lambda constant domain. The three HC-CDRs comprise SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and the three LC-CDRs comprise SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

In further embodiments, the effector-silent anti-CTLA-4 antibody comprises either (i) the VH and VL domains of ipilimumab, (ii) the VH and VL domains of tremelimumab, (iii) the VH and VL domains of REGN4659, (iv) the VH and V domains of AGEN1884w, or (v) the V _H and VL domains of anti-CTLA-4 antibody clone 2C8 disclosed in International Patent Application WO2017194265. The ipilimumab VH domain comprises the amino acid sequence set forth in SEQ ID NO:7 and VL domain comprises the amino acid sequence set forth in SEQ ID NO: 8. The tremelimumab VH domain comprises the amino acid sequence set forth in SEQ ID NO: 15 and VL domain comprises the amino acid sequence set forth in SEQ ID NO: 16. The REGN4659 VH domain comprises the amino acid sequence set forth in SEQ ID NO:95 and VL domain comprises the amino acid sequence set forth in SEQ ID NO:96. The AGEN1884w VH domain comprises the amino acid sequence set forth in SEQ ID NO:97 and VL domain comprises the amino acid sequence set forth in SEQ ID NO:98. In particular embodiments, the VH and VL domains further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In further embodiments, the effector-silent anti-CTLA-4 antibody comprises the VH and VL domains of 8D2/8D2 (RE) (See U.S. Published Patent Application No.

20170216433 and International Application WO2018183408), 8D2H1L1, 8D2H2L2, 8D3H3L3, 8D2H2L15, or 8D2H2L17, wherein the VH domain is fused or linked to an HC constant domain that displays no measurable binding to the FcyRIIIA, FcyRIIA, and FcyRI or reduced binding compared to a polypeptide comprising the wild-type IgG constant domain region as determined by a Biacore assay and the VL domain is fused or linked to a LC kappa or lambda constant domain.

In particular embodiments, the effector-silent anti-CTLA-4 antibody comprises a variant of 8D2/8D2 (RE), 8D2H1L1, 8D2H2L2, 8D2H2L15, or 8D2H2L17, wherein the methionine at position 18 in the VJJ amino acid sequence of the variant is substituted with isoleucine. Thus, the effector-silent anti-CTLA-4 antibody may comprise the VH and VL of 8D2/8D2 (In variant 1, the VH and VL of 8D2H1L1 -Variant 1, the VH and VL of 8D2H2L2 -Variant 1, the V _H and V _L of 8D2H2L 15-Variant 1, or the V _H and V _L of 8D2H2L 17-Variant 1.

In further embodiments, the effector silent anti-CTLA4 antibody has a (i) a VH domain comprising the amino acid sequence set forth in SEQ ID NO:73 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:74; (ii) a VH domain comprising the amino acid sequence set forth in SEQ ID NO:75 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:76; (iii) a VH domain comprising the amino acid sequence set forth in SEQ ID NO:77 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:78; (iv) a V _]-[ domain comprising the amino acid sequence set forth in SEQ ID NO:79 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 80; (v) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO: 81 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 82; (vi) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:83 and a V domain comprising the amino acid sequence set forth in SEQ ID NO: 84; (vii) aVp domain comprising the amino acid sequence set forth in SEQ ID NO: 85 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 86; (viii) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO: 87 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 88; (ix) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO: 89 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:90; (x) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:91 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:92; or (xi) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:93 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:94. In particular embodiments, the V _j q and VL domains further comprise 1, 2, 3, 4,

5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In further embodiments of the effector-silent anti-CTLA-4 antibody, the V _j q domain is fused or linked to an IgGq HC constant domain or an IgG _| . IgG2, or IgGq HC constant domain that has been modified to include one or more mutations to render the resulting anti-CTLA4 antibody effecter-silent.

In one embodiment, the effector-silent anti-CTLA-4 antibody comprises an IgG _j Fc domain having (i) a mutation in the V-glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes /V-glycosylation at said V-glycosylation site or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; (ii) an amino acid substitution mutation selected from the group consisting of N297A, L234A/L235A/D265A, L234A/L235A/P329G, L235E, D265A,

E233A/L235A,S267E/L328F, S2339D/A330L/I332E, L235G/G236R, N297A/D356E/L358M, L234F/L235E/P331 S/D365E/L358M, and D265A/N297G or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; or (iii) a mutation in the /V-glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes /V-glycosylation at said /V-glycosylation site and an amino acid substitution mutation selected from the group consisting of L234A/L235A/D265A,

L234A/L235A/P329G, L235E, D265A, E233A/L235A,S267E/L328F, S2339D/A330L/I332E, L235G/G236R, D356E/L358M, L234F/L235E/P331S/D365E/L358M, and D265A or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

In another embodiment, the effector-silent anti-CTLA-4 antibody comprises an IgG2 Fc domain having (i) a mutation in the A-glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes A-glycosylation at said A-glycosylation site or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; (ii) an amino acid substitution mutation selected from the group consisting of N297A/D265S, D265A, P329G/D265A/N297G, or

V234A/G237A/P238S/H268A/V309L/A330S/P331S or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; or (iii) a mutation in the A-glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes X-glycosylation at said X-glycosylation site and an amino acid substitution mutation selected from the group consisting of N297A/D265S, D265A,

P329G/D265A/N297G, or V234A/G237A/P238S/H268A/V309L/A330S/P331S or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

In a further embodiment, the effector-silent anti-CTLA-4 antibody comprises an IgG4 Fc domain having an S228P amino acid substitution and further comprising (i) a mutation in the X-glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes X-glycosylation at said X-glycosylation site or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; (ii) an amino acid substitution mutation selected from the group consisting of N267A, P329G, and D265A/N297A or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions; or (iii) a mutation in the N- glycosylation site Asn-Xaa-Ser/Thr beginning at amino acid position 297 that abolishes N- glycosylation at said X-glycosylation site and an amino acid substitution mutation selected from the group consisting of N267A, P329G, and D265A/N297A or the mutated Fc domain further comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein the amino acid positions in (i), (ii), and (iii) are identified according to Eu numbering.

Tables 4-18 provide specific exemplary anti-CTLA-4 antibodies that may be used in combination with an anti-PD-1 or anti-PD-Ll antibody in a therapy to treat an individual who has cancer. The present invention also provides the antibodies shown in the tables except for ipilimumab consisting solely of an N297A substitution and compositions, each composition comprising an antibody shown in the tables and a pharmaceutically acceptable carrier except for a composition comprising ipilimumab consisting solely of an N297A substitution. All HC amino acid substitution positions in Tables 4-18 are according to the Eu numbering scheme.

(c) Exemplary effector -silent anti-CTLA-4 antibody fragments

Exemplary effector-silent anti-CTLA-4 antibody fragments that may be used in the combination therapy of the present invention and compositions comprising the same include any antibody fragment that binds CTLA-4 and inhibits CTLA-4 from binding B7. Specific examples of these anti-CTLA-4 antibody fragments include the following anti-CTLA-4 antibody fragments and compositions, each composition comprising an effector-silent anti-CTLA-4 antibody fragment and a pharmaceutically acceptable carrier. In particular embodiments, the effector-silent anti-CTLA-4 antibody fragment is an Fv, scFv, F(ab), or F(ab’)2 that comprises (i) a VH comprising the three HC-CDRs of ipilimumab and (ii) a VL comprising the three LC-CDRs of ipilimumab. The three HC-CDRs comprise SEQ ID NO: l, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the three LC-CDRs comprise SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:7, respectively.

In particular embodiments, the effector-silent anti-CTLA-4 antibody fragment comprises (i) a VH comprising the three HC-CDRs of tremelimumab and (ii) a VL comprising the three LC-CDRs of tremelimumab. The three HC-CDRs comprise SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively, and the three LC-CDRs comprise SEQ ID NO: 12,

SEQ ID NO: 13, and SEQ ID NO: 14, respectively.

In particular embodiments, the effector-silent anti-CTLA-4 antibody fragment comprises either (i) the VH and V domains of ipilimumab, (ii) the VH and VL domains of tremelimumab, (iii) the VH and VL domains of REGN4659, (iv) the VH and VL domains of AGEN1884w, or (v) the VH and VL domains of anti-CTLA-4 antibody clone 2C8 disclosed in International Patent Application WO2017194265. The ipilimumab VH domain comprises the amino acid sequence set forth in SEQ ID NO:7 and VL domain comprising the amino acid sequence set forth in SEQ ID NO: 8. The tremelimumab VH domain comprises the amino acid sequence set forth in SEQ ID NO: 15 and VL domain comprising the amino acid sequence set forth in SEQ ID NO: 16. The REGN4659 VH domain comprises the amino acid sequence set forth in SEQ ID NO: 95 and VL domain comprising the amino acid sequence set forth in SEQ ID NO: 96. The AGEN1884w VH domain comprises the amino acid sequence set forth in SEQ ID NO:97 and VL domain comprising the amino acid sequence set forth in SEQ ID NO:98.

In particular embodiments, the effector-silent anti-CTLA-4 antibody fragment comprises the VH and VL of ipilimumab, the VH and VL of tremelimumab, the VH and VL of REGN4659, the V _H and V _L of AGEN1884w, the V _H and V _L of 8D2/8D2 (RE), the V _H and V _L of 8D2H1L1, the V _H and V _L of 8D2H2L2, the V _H and V _L of 8D3H3L3, the V _H and V _L of 8D2H2L15, or the V _H and V _L of 8D2H2L17.

In particular embodiments, the anti-CTLA-4 antibody or anti-CTLA-4 antibody fragment comprises the VH and VL of 8D2/8D2 (RE)-Variant 1, the VH and VL of 8D2H1L1- Variant 1, the VH and VL of 8D2H2L2-Variant 1, the VH and VL of 8D2H2L 15 -Variant 1, or the V _H and V _L of 8D2H2L17-Variant 1.

In particular embodiments, the effector-silent anti-CTLA-4 antibody fragment comprises either (i) a VH domain comprising the amino acid sequence set forth in SEQ ID

NO:73 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:74; (ii) a VH domain comprising the amino acid sequence set forth in SEQ ID NO:75 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:76; (iii) a VH domain comprising the amino acid sequence set forth in SEQ ID NO:77 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:78; (iv) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:79 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 80; (v) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO: 81 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 82; (vi) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:83 and a V domain comprising the amino acid sequence set forth in SEQ ID NO: 84; (vii) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO: 85 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 86; (viii) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:87 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 88; (ix) aVp domain comprising the amino acid sequence set forth in SEQ ID NO: 89 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:90; (x) a Vq domain comprising the amino acid sequence set forth in SEQ ID NO:91 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:92; or (xi) a V _j q domain comprising the amino acid sequence set forth in SEQ ID NO:93 and a VL domain comprising the amino acid sequence set forth in SEQ ID NO:94.

In particular embodiments, the effector-silent anti-CTLA-4 antibody fragment comprises one or more immunoglobulin single variable domains (ISVDs), each ISVD comprising the variable domain (VHH) of a camelid heavy chain only antibody with the proviso that the ISVD does not comprise a CDR1 comprising the amino sequence FYGMG (SEQ ID NO:69, a CDR2 comprising the amino acid sequence DIRTS AGRTTYADSVKG (SEQ ID NO:70), and a CDR3 comprising amino acid EMSGISGWDY (SEQ ID NO:71) or

EPSGISGWDY (SEQ ID NO:72) as those ISVDs disclosed in International Patent Application W02008071447, WO2017087587, and WO2017087588 and ISVD variants comprising 1, 2, or 3 mutations in CDR3 as disclosed in W02008071447, with the exception that not excluded by the proviso are ISVDs comprising said CDRs in embodiments wherein the one or more ISVDs are fused or linked to an effector-silent antibody constant domain or Fc domain, for example, any one of the effector-silent antibody constants or Fc domains disclosed herein.

(d) Exemplary anti-PD-1 antibodies

Exemplary anti-PD-1 antibodies that may be used in the combination therapy of the present invention include any antibody that binds PD-1 and inhibits PD-1 from binding PD- Ll. In a further embodiment, the exemplary anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and cemiplimab-rwlc. Exemplary antibodies include the following anti-PD-1 antibodies and compositions comprising an anti-PDl antibody and a pharmaceutically acceptable salt.

Pembrolizumab, also known as KEYTRUDA, lambrolizumab, MK-3475 or SCH- 900475, is a humanized anti-PD-1 antibody described in U.S. Pat. No. 8,354,509 and W02009/114335 and disclosed, e.g., in Hamid, et al, New England J. Med. 369 (2): 134-144 (2013). The heavy and light chains for pembrolizumab are shown by the amino acid sequences set forth in SEQ ID NOs: 27and 28, respectively.

Nivolumab, also known as OPDIVO, MDX-1106-04, ONO-4538, or BMS- 936558, is a fully human IgG4 anti-PD-1 antibody described in W02006/121168 and U.S. Pat. No. 8,008,449. The heavy and light chains for nivolumab are shown by the amino acid sequences set forth in SEQ ID NOs: 25and 26, respectively.

Cemiplimab-rwlc, also known as cemiplimab, LIBTAYO or REGN2810, is a recombinant human IgGq monoclonal antibody that is described in WO2015112800 and U.S.

Pat. No. 9,987,500. The heavy and light chains for cemiplimab-rwlc are shown by the amino acid sequences set forth in SEQ ID NOs: 101 and 102, respectively.

In particular embodiments, the anti-PD-1 antibody comprises (i) aVp comprising the three HC-CDRs of pembrolizumab fused or linked to an effector-silent HC constant domain and (ii) a VL comprising the three LC-CDRs of pembrolizumab fused or linked to a LC kappa or lambda constant domain. The three HC-CDRs comprise SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively, and the three LC-CDRs comprise SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively.

In particular embodiments, the anti-PD-1 antibody comprises (i) aVp comprising the three HC-CDRs of nivolumab fused or linked to an effector-silent HC constant domain and (ii) a VL comprising the three LC-CDRs of nivolumab fused or linked to a LC kappa or lambda constant domain. The three HC-CDRs comprise SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively, and the three LC-CDRs comprise SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:2, respectively.

In particular embodiments, the anti-PD-1 antibody comprises (i) aVp comprising the three HC-CDRs of cemiplimab-rwlc fused or linked to an effector-silent HC constant domain and (ii) a V comprising the three LC-CDRs of nivolumab fused or linked to a LC kappa or lambda constant domain.

In particular embodiments, the anti-PD-1 antibody comprises (i) the V _j q and VL domains of pembrolizumab, wherein the V _j q domain is fused or linked to an effector-silent HC constant domain and the VL domain is fused or linked to a LC kappa or lambda constant domain; (ii) the V _j q and VL domains of nivolumab, wherein the V _j q domain is fused or linked to an effector-silent HC constant domain and the VL domain is fused or linked to an LC kappa or lambda constant domain; or (iii) the V _j q and VL domains of cemiplimab-rwlc, wherein the V _j q domain is fused or linked to an effector-silent HC constant domain and the VL domain is fused or linked to an LC kappa or lambda constant domain. The pembrolizumab V _j q domain comprises the amino acid sequence set forth in SEQ ID NO:29 and the VL domain comprises the amino acid sequence set forth in SEQ ID NO:30. The nivolumab V _j q domain comprises the amino acid sequence set forth in SEQ ID NO:23 and the VL domain comprises the amino acid sequence set forth in SEQ ID NO:24. The cemiplimab-rwlc V _j q domain comprises the amino acid sequence set forth in SEQ ID NO:99 and VL domain comprises the amino acid sequence set forth in SEQ ID NO: 100. In particular embodiments, the V _j q and V domains may further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In particular embodiments, the anti-PD-1 antibody V _j q domain may be fused or linked to an IgG _j , IgG2, IgG3, or IgGq HC constant domain that is not currently linked to the particular V _j q or is linked to an IgG _| . IgG2, IgG3, or IgGq HC constant domain has been modified to include one or more mutations in the Fc domain that render the resulting anti-PD-1 antibody effecter-silent.

In certain embodiments, the HC constant domain is of an IgG _j , IgG2, IgG3, or IgGq isotype, which is modified to lack /V-glycosylation of the asparagine (Asn) residue at position 297 of the HC constant domain by replacing the codon for the Asn at position 297 in the nucleic acid molecule encoding the HC constant domain with a codon for another amino acid, for example Gin. In further embodiments, such IgGs modified to lack /V-glycosylation at position 297 further includes one or more additional mutations disclosed herein for eliminating detectable effector function. In particular embodiments, the HC constant domain is a human HC constant domain. In particular embodiments, the molecules further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In particular embodiments, the present invention provides an anti-PD-1 antibody that comprises an IgGq HC constant domain that has been modified to have an S228P substitution and further include in addition to the S228P substitution, a P239G, D265A, or D265A/N297G amino acid substitutions, wherein the positions are identified according to Eu numbering. In particular embodiments of the above, the IgGq HC constant domain is a human

HC constant domain. In particular embodiments, the molecules further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In another embodiment, the anti-PD-1 antibody may comprise a human IgG _j isotype, in which the Fc domain of the HC constant domain has been modified to be effector- silent by substituting the amino acids from position 233 to position 236 of the IgG _j with the corresponding amino acids of the human IgG2 HC and substituting the amino acids at positions 327, 330, and 331 with the corresponding amino acids of the human IgGq HC, wherein the positions are identified according to Eu numbering. In particular embodiments, the HC molecules further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non conservative mutations.

In another embodiment, the Fc domain of the IgG _] IgG2, IgG3, or IgG4 HC constant domain is modified to include one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S and wherein said polypeptide exhibits no measurable binding to the FcyRIIIA, FcyRIIA, and FcyRI or reduced binding compared to a polypeptide comprising the wild-type IgG constant domain region as determined by a Biacore assay. These and other substitutions are disclosed in WO9428027; W02004099249; W020121300831, U.S. Pat. Nos. 9,708,406; 8,969,526; 9,296,815;

Sondermann et al. Nature 406, 267-273 (20 Jul. 2000)). In particular embodiments, the HC molecules further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non conservative mutations.

Tables 19-27 provide specific exemplary anti -PD- 1 antibodies that may be used in combination with an anti-CTLA-4 antibody as disclosed herein in a therapy to treat an individual who has cancer. The present invention also provides the antibodies shown in the tables and compositions, each composition comprising an antibody shown the tables and a pharmaceutically acceptable carrier. All HC amino acid substitution positions in Tables 19-27 are according to the Eu numbering scheme.

(e) Exemplary anti-PD-1 antibody fragments

Exemplary anti-PD-1 antibody fragments that may be used in the combination therapy of the present invention include any anti-PD-1 antibody fragment that binds PD-1 and inhibits PD-1 from binding PD-L1 and further include the following anti-PD-1 antibody fragments that bind PD-1 and compositions comprising the following anti-PD-1 antibody fragments and a pharmaceutically acceptable carrier.

In particular embodiments, the antibody fragment is an Fv or scFv comprising the pembrolizumab V _j-[ having the amino acid sequence set forth in SEQ ID NO:29 and the pembrolizumab VL having the amino acid sequence set forth in SEQ ID NO:30.

In particular embodiments, the anti-PD-1 antibody fragment is a F(ab) comprising the pembrolizumab V _j q having the amino acid sequence set forth in SEQ ID NO:29 and the pembrolizumab V _j q having the amino acid sequence set forth in SEQ ID NO:30.

In particular embodiments, the anti-PD-1 antibody fragment is a F(ab’)2 comprising the pembrolizumab V _j q having the amino acid sequence set forth in SEQ ID NO:29 and the pembrolizumab VH having the amino acid sequence set forth in SEQ ID NO:30.

In particular embodiments, the anti-PD-1 antibody fragment is an Fv or scFv comprising the nivolumab V _j q having the amino acid sequence set forth in SEQ ID NO:23 and the nivolumab V _j q having the amino acid sequence set forth in SEQ ID NO:24.

In particular embodiments, the anti-PD-1 antibody fragment is a F(ab) comprising the nivolumab VH having the amino acid sequence set forth in SEQ ID NO:23 and the nivolumab V _j q having the amino acid sequence set forth in SEQ ID NO:24. In particular embodiments, the anti-PD-1 antibody fragment is a F(ab’)2 comprising the nivolumab V _j-[ having the amino acid sequence set forth in SEQ ID NO:23 and the nivolumab V _j q having the amino acid sequence set forth in SEQ ID NO:24.

In particular embodiments, the anti-PD-1 antibody fragment comprises one or more immunoglobulin single variable domains (ISVDs), each ISVD comprising the variable domain (VHH) °f ^a camelid heavy chain only antibody with the proviso that ISVD does not comprise a CDR1 comprising the amino sequence THAMG (SEQ ID NO:73, a CDR2 comprising the amino acid sequence VITWSGGITTYADSVKG (SEQ ID NO:74) or

VITV S GGITYY AD S VKG (SEQ ID NO:75), and a CDR3 comprising amino acid

DKHQSSWYDY (SEQ ID NO:76) or DKHQSSFYDY (SEQ ID NO:77) as those ISVDs disclosed in International Patent Application W02008071447, WO2017087587, and

WO2017087589 and variants comprising 1, 2, or 3 mutations in CDR3 as set forth in

W02008071447, with the exception that not excluded by the proviso are ISVDs comprising said CDRs in embodiments wherein the one or more ISVDs are fused or linked to an effector-silent antibody constant domain or Fc domain, for example, any one of the effector-silent antibody constant domains or Fc domains disclosed herein.

(f) Exemplary anti-PD-Ll antibodies

Exemplary anti-PD-Ll antibodies that may be used in the combination therapy of the present invention include any anti-PD-Ll antibody that inhibits PD-1 from binding PD-L1 and further includes the following anti-PD-Ll antibodies and compositions comprising the following anti-PD-Ll antibodies and a pharmaceutically acceptable carrier. In particular embodiments, the anti-PD-Ll antibody is selected from the group consisting atezolizumab, avelumab, and durvalumab.

In particular embodiments, the anti-PD-Ll antibody comprises (i) the V _j q and VL domains of atezolizumab, wherein the V _j q domain is fused or linked to an HC constant domain or effector-silent HC constant domain and the VL domain is fused or linked to an LC kappa or lambda constant domain, (ii) the V _j q and V domains of avelumab, wherein the V _j q domain is fused or linked to an HC constant domain or effector-silent HC constant domain and the VL domain is fused or linked to an LC kappa or lambda constant domain, or (iii) the V _j q and VL domains of durvalumab, wherein the V _j q domain is fused or linked to an HC constant domain or effector-silent HC constant domain and the VL domain is fused or linked to an LC kappa or lambda constant domain. The durvalumab V _j q domain comprises the amino acid sequence set forth in SEQ ID NO: 103 and VL domain comprises the amino acid sequence set forth in SEQ ID NO: 104. The avelumab V _j q domain comprises the amino acid sequence set forth in SEQ ID NO: 105 and VL domain comprises the amino acid sequence set forth in SEQ ID NO: 106. The atezolizumab V _j q domain comprises the amino acid sequence set forth in SEQ ID NO: 107 and VL domain comprises the amino acid sequence set forth in SEQ ID NO: 108. In particular embodiments, the V _j q and VL domains further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In particular embodiments, the anti-PD-Ll antibody V _j q domain may be fused or linked to an IgG _j , IgG2, IgG3, or IgGq HC constant domain that is not currently linked to the particular V _j q or is linked to an IgG p IgG2, IgG3, or IgGq HC constant domain has been modified to include one or more mutations in the Fc domain that render the resulting anti-PD-Ll antibody effecter-silent.

In certain embodiments, the HC constant domain is of the IgG _j , IgG2, IgG3, or IgGq isotype, which is modified to lack /V-glycosylation of the asparagine (Asn) residue at position 297 of the HC constant domain by replacing the codon for the Asn at position 297 in the nucleic acid molecule encoding the HC constant domain with a codon for another amino acid, for example Gin. Alternatively, the codon for Ser may be replaced with the codon for Pro or the codon for Thr may be replaced with any codon except the codon for Ser, e.g. N297A, N297G, or N297D. Alternatively, all three codons are modified. In further embodiments, such IgGs modified to lack /V-glycosylation at position 297 further includes one or more additional mutations disclosed herein for eliminating detectable effector function. In particular embodiments, the HC constant domain is a human HC constant domain. In particular embodiments, the molecules further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In particular embodiments, the present invention provides an anti-PD-Ll antibody that comprises an IgGq HC constant domain that has been modified to have an S228P substitution and further include in addition to the S228P substitution, a P239G, D265A, or D265A/N297G amino acid substitutions, wherein the positions are identified according to Eu numbering. In particular embodiments of the above, the IgGq HC constant domain is a human

HC constant domain. In particular embodiments, the molecules further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non-conservative mutations.

In another embodiment, the anti-PD-Ll antibody may comprise a human IgG _j isotype, in which the Fc domain of the HC constant domain has been modified to be effector- silent by substituting the amino acids from position 233 to position 236 of the IgG _j with the corresponding amino acids of the human IgG2 HC and substituting the amino acids at positions 327, 330, and 331 with the corresponding amino acids of the human IgGq HC, wherein the positions are identified according to Eu numbering. In particular embodiments, the HC molecules further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non conservative mutations.

In another embodiment, the Fc domain of the IgG _] IgG2, IgG3, or IgG4 HC constant domain is modified to include one or more amino acid substitutions selected from E233P, L234A, L235A, L235E, N297A, N297D, D265S, and P331S and wherein said polypeptide exhibits no measurable binding to the FcyRIIIA, FcyRIIA, and FcyRI or reduced binding compared to a polypeptide comprising the wild-type IgG constant domain region as determined by a Biacore assay. These and other substitutions are disclosed in WO9428027; W02004099249; W020121300831, U.S. Pat. Nos. 9,708,406; 8,969,526; 9,296,815;

Sondermann et al. Nature 406, 267-273 (20 Jul. 2000)). In particular embodiments, the HC molecules further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions, insertions, and/or deletions, wherein said substitutions may be conservative mutations or non conservative mutations.

Tables 28-36 provide exemplary anti-PD-Ll antibodies, which may be used in combination with an anti-CTLA-4 antibody as disclosed herein in a therapy to treat an individual who has cancer. The present invention also provides the antibodies shown the tables, except for antibodies 25-9 and 31-8, and compositions, each composition comprising an antibody shown in the table, except for antibodies 25-9 and 31-8, and a pharmaceutically acceptable carrier. All HC amino acid substitution positions in Tables 28-36 are according to the Eu numbering scheme.

(g) Exemplary anti-PD-Ll antibody fragments

Exemplary anti-PD-Ll antibody fragments that may be used in the combination therapy of the present invention includes any anti-PD-Ll antibody fragment that binds PD-L1 and inhibits PD-L1 from binding PD- land further includes the following anti-PD-Ll antibody fragments and compositions, each composition comprising a following anti-PD-Ll antibody fragment and a pharmaceutically acceptable carrier.

In particular embodiments, the anti-PD-Ll antibody fragment is an Fv or scFv comprising the durvalumab V _j-[ having the amino acid sequence set forth in SEQ ID NO: 103 and the durvalumab VL having the amino acid sequence set forth in SEQ ID NO: 104.

In particular embodiments, the anti-PD-Ll antibody fragment is a F(ab) comprising the durvalumab V _j q having the amino acid sequence set forth in SEQ ID NO: 103 and the durvalumab V _j q having the amino acid sequence set forth in SEQ ID NO: 104.

In particular embodiments, the anti-PD-Ll antibody fragment is a F(ab’)2 comprising the durvalumab V _j q having the amino acid sequence set forth in SEQ ID NO: 103 and the durvalumab VH having the amino acid sequence set forth in SEQ ID NO: 104.

In particular embodiments, the anti-PD-Ll antibody fragment is an Fv or scFv comprising the avelumab V _j q having the amino acid sequence set forth in SEQ ID NO: 105 and the avelumab V _j q having the amino acid sequence set forth in SEQ ID NO: 106.

In particular embodiments, the anti-PD-Ll antibody fragment is a F(ab) comprising the avelumab VH having the amino acid sequence set forth in SEQ ID NO: 105 and the avelumab V _j q having the amino acid sequence set forth in SEQ ID NO: 106. In particular embodiments, the anti-PD-Ll antibody fragment is a F(ab’)2 comprising the avelumab V _j-[ having the amino acid sequence set forth in SEQ ID NO: 105 and the avelumab V _j q having the amino acid sequence set forth in SEQ ID NO: 106.

In particular embodiments, the anti-PD-Ll antibody fragment is an Fv or scFv comprising the atezolizumab V _j q having the amino acid sequence set forth in SEQ ID NO: 107 and the atezolizumab V _j q having the amino acid sequence set forth in SEQ ID NO: 108.

In particular embodiments, the anti-PD-Ll antibody fragment is a F(ab) comprising the atezolizumab VH having the amino acid sequence set forth in SEQ ID NO: 107 and the atezolizumab V _j q having the amino acid sequence set forth in SEQ ID NO: 108.

In particular embodiments, the anti-PD-Ll antibody fragment is a F(ab’)2 comprising the atezolizumab V _j q having the amino acid sequence set forth in SEQ ID NO: 107 and the atezolizumab V _j q having the amino acid sequence set forth in SEQ ID NO: 108.

In particular embodiments, the anti-PD-Ll antibody fragment comprises one or more immunoglobulin single variable domains (ISVDs), each ISVD comprising the variable domain (VHH) °f ^a camelid heavy chain only antibody with the proviso that ISVD does not comprise an anti-PD-Ll ISVD disclosed in International Application W02008071447 having SEQ ID NO: 394-399 therein or disclosed in W02009030285, both of which are incorporated herein by reference, with the exception that not excluded by the proviso are ISVDs wherein the one or more ISVDs are fused or linked to an effector-silent antibody constant domain or Fc domain, for example, any one of the effector-silent antibody constant domains or Fc domains disclosed herein.

(h) Exemplary combination therapy dosing regimens

The present invention provides anti-cancer therapies that combine the immune- stimulating effects of a PD-1 blocking agent with the anti -tumor effects of a CTLA-4 blocking agent but without the dermatologic or gut irAEs typically observed for CTLA-4 blocking agents administered in combination with PD-1 blocking agents. A feature of the present invention is that the CTLA-4 blocking agent lacks measurable binding to one or more FcRs as determined in a Biacore assay or reduced binding to one or more FcRs compared to that of a wild-type antibody of the same isotype as measured in a Biacore assay. Thus, the CTLA-4 blocking agents display no measurable or display reduced effector function, which enables the effector-silent CTLA-4 blocking agents to be used in combination therapies with PD-1 blocking agents at doses and dosing durations not available with CTLA-4 blocking agents that display effector function. This feature distinguishes the CTLA-4 blocking agents of the present invention from the currently available CTLA-4 blocking agents.

In a typical dosing regimen of the present invention, the CTLA-4 blocking agent and the PD-1 blocking agent may be administered to the individual concurrently in separate doses and in different formats. In general, the CTLA-4 blocking agent of the present invention may be administered in a combination therapy with a PD-1 blocking agent at least at the same dose, dosing frequency, and treatment duration currently approved by the U.S. FDA for the ipilimumab/nivolumab combination therapy for particular indications. However, the combination therapy is not limited to the particular indications approved by the U.S. FDA but may include any indication that may benefit from the combination therapy of the present invention. The currently approved dose is 1 mg/kg of ipilimumab following the administration of nivolumab provided at a dose of 3 mg/kg. This dose combination may then be repeated every three weeks for four doses with the doses of nivolumab continuing every two weeks thereafter as needed. However, in further embodiments, the CTLA-4 blocking agent of the present invention may be administered in the combination therapy at a dose that is more than 1 mg/kg, for example a dose of at least 3 mg/kg. In a further still embodiment, the dose may be at least 10 mg/kg and in further still embodiments, the dose may be between about 1 mg/kg and 10 mg/kg. In particular embodiments, the CTLA-4 blocking agent of the present invention may be administered for at the same dosing frequency and treatment duration as that in the approved ipilimumab/nivolumab combination therapy. In particular embodiments, the CTLA-4 blocking agents of the present invention may be administered at the same dosing frequency and treatment duration as that for nivolumab in the approved ipilimumab/nivolumab combination therapy.

In particular embodiments of the combination therapy, the CTLA-4 blocking agent is administered in a dose that is not based on the weight of the individual. Thus, in particular embodiments, the CTLA-4 binding agent may be administered at a dose between about 10 mg and 300 mg. In a further embodiment, the dose is selected from the group consisting of 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, and 300 mg.

In the combination therapy of the present invention, the PD-1 blocking agent may be administered at the same dose, dosing frequency, and treatment duration as that approved for the PD-1 blocking agent in a monotherapy for particular indications. The dose of the CTLA-4 blocking agent may be as cited above and the CTLA-4 blocking agent may be administered at the same dosing frequency and treatment duration as cited above or at a dosing frequency and treatment duration as for the particular PD-1 blocking agent that is paired with the CTLA-4 blocking agent.

The particular dose of the currently marketed PD-1 blocking agents vary between the PD-1 blocking agents, thus in particular embodiments of the combination therapy of the present invention, the dose, dosing frequency, and/or treatment duration may be at least the same as that approved by the U.S. FDA for the particular PD-1 blocking agent for particular indications. For example, pembrolizumab is approved for a dose of 200 mg every three weeks as needed (pediatric individuals (two years up to 18 years) at 2 mg/kg up to 200 mg every three weeks as needed); nivolumab is approved at a dose of 3 mg/kg every 2 weeks; cemiplimab-rwlc is approved for a dose of 350 mg every three weeks as needed; atezolizumab is approved for a dose of 1200 mg every three weeks as needed; avelumab is approved for a dose of 10 mg/kg or 800 mg every two weeks as needed; and durvalumab is approved for a dose of 10 mg/kg every two weeks as needed.

In particular embodiments of the combination therapy, the PD-1 blocking agent is an anti-PD-1 antibody or anti-PD-1 antibody fragment, which may be administered at a dose from about 150 mg to about 250 mg, from about 175 mg to about 250 mg, from about 200 mg to about 250 mg, from about 150 mg to about 240 mg, from about 175 mg to about 240 mg, or from about 200 mg to about 240 mg. In some embodiments, the dose of the anti-PD-1 antibody or antigen binding fragment thereof is 150 mg, 175 mg, 200 mg, 225 mg, 240 mg, or 250 mg. In further embodiments, the anti-PD-1 antibody or anti-PD-1 antibody fragment may be administered at a frequency of every three weeks as needed. In another embodiment of the combination therapy of the present invention, the anti-PD-1 antibody or anti-PD-1 antibody fragment may be administered at dose greater than 250 mg, for example, a dose of about 400 mg at a frequency of every six weeks as needed.

In particular embodiments of the combination therapy, the PD-1 blocking agent is an anti-PD-1 antibody or anti-PD-1 antibody fragment, which may be administered at a dose from about 10 mg/kg to about 1200 mg. In further embodiments, the anti-PD-1 antibody or anti- PD-1 antibody fragment may be administered at a frequency of every two to three weeks as needed.

While the PD-1 blocking agent may be administered at least at the doses, dosing frequencies, and treatment durations approved for the currently marketed PD-1 blocking agents in a monotherapy, the actual doses, dosing frequencies, and treatment durations for any particular combination of the present invention may differ from those that are approved for the PD-1 blocking agent monotherapies. Thus, in particular embodiments of the combination therapy of the present invention, the dose, dosing frequency, and treatment duration of any particular PD-1 blocking agent in the combination therapy will be determined from clinical trials conducted for the combination therapy.

In a particular embodiment of the combination therapy, the PD-1 blocking agent is nivolumab or an effector-silent variant of nivolumab, which is administered to an individual intravenously at a dose of 3 mg/kg over 30 to 60 minutes every two-three weeks as needed and wherein each dose of the CTLA-4 blocking agent is administered intravenously following the administration of the PD-1 blocking agent for the same treatment duration as the PD-1 blocking agent or for duration less than or more than the PD-1 blocking agent duration. In a particular embodiment, the nivolumab or effector-silent variant of nivolumab is administered intravenously to an individual at an initial dose of 3 mg/kg intravenously over 30 minutes followed by administration of the CTLA-4 blocking agent intravenously over 30 minutes on the same day, every three weeks for four doses, then nivolumab is administered intravenously at a fixed dose of 240 mg every two weeks over 30 minutes or 480 mg every four weeks over 30 minutes.

In a particular embodiments, the PD-1 blocking agent is pembrolizumab or effector-silent variant of pembrolizumab, which is administered to an adult individual intravenously at a dose of 200 mg over 30 minutes every three weeks as needed or to a pediatric individual intravenously at a dose of 2 mg/kg up to a maximum of about 200 mg over 30 minutes every three weeks wherein each treatment is followed by a dose of the CTLA-4 blocking agent wherein each dose of the CTLA-4 blocking agent is administered intravenously following administration of the PD-1 blocking agent for the same treatment duration as the PD-1 blocking agent or for duration less than or more than the PD-1 blocking agent duration.

In a particular embodiments, the PD-1 blocking agent is pembrolizumab or effector-silent variant of pembrolizumab, which is administered to an adult individual intravenously at a dose of 400 mg over 30 minutes every six weeks as needed wherein each treatment is followed by a dose of the CTLA-4 blocking agent wherein each dose of the CTLA-4 blocking agent is administered intravenously following the administration of the PD-1 blocking agent for the same treatment duration as the PD-1 blocking agent or for duration less than or more than the PD-1 blocking agent duration.

In a particular embodiment of the combination therapy, the PD-1 blocking agent is cemiplimab-rwlc or an effector-silent variant of cemiplimab-rwlc, which is administered to an individual intravenously at a dose of 350 mg over 30 minutes every three weeks as needed and wherein each dose of the CTLA-4 blocking agent is administered intravenously following the administration of the PD-1 blocking agent for the same treatment duration as the PD-1 blocking agent or for duration less than or more than the PD-1 blocking agent duration. In a particular embodiment, the cemiplimab-rwlc or effector-silent variant of cemiplimab-rwlc is administered intravenously to an individual at an initial dose of 350 mg over 30 minutes followed by administration of the CTLA-4 blocking agent over 30 minutes on the same day every three weeks as needed.

In a particular embodiment of the combination therapy, the PD-1 blocking agent is atezolizumab or an effector-silent variant of atezolizumab, which is administered to an individual intravenously at a dose of 1200 mg over 60 minutes every three weeks as needed and wherein each dose of the CTLA-4 blocking agent is administered intravenously following the administration of the PD-1 blocking agent for the same treatment duration as the PD-1 blocking agent or for duration less than or more than the PD-1 blocking agent duration. In a particular embodiment, the atezolizumab or effector-silent variant of atezolizumab is administered intravenously to an individual at an initial dose of 1200 mg over 60 minutes followed by administration of the CTLA-4 blocking agent over 30 minutes on the same day every three weeks as needed. In a particular embodiment of the combination therapy, the PD-1 blocking agent is avelumab or an effector-silent variant of avelumab, which is administered to an individual intravenously at a dose of 10 mg/kg or 800 mg over 60 minutes every two weeks as needed and wherein each dose of the CTLA-4 blocking agent is administered intravenously following the administration of the PD-1 blocking agent for the same treatment duration as the PD-1 blocking agent or for duration less than or more than the PD-1 blocking agent duration. In a particular embodiment, the avelumab or effector-silent variant of avelumab is administered intravenously to an individual at an initial dose of 10 mg/kg or 800 mg over 60 minutes followed by administration of the CTLA-4 blocking agent over 30 minutes on the same day every two weeks as needed.

In a particular embodiment of the combination therapy, the PD-1 blocking agent is durvalumab or an effector-silent variant of durvalumab, which is administered to an individual intravenously at a dose of 10 mg/kg over 60 minutes every two weeks as needed and wherein each dose of the CTLA-4 blocking agent is administered intravenously following the

administration of the PD-1 blocking agent for the same treatment duration as the PD-1 blocking agent or for duration less than or more than the PD-1 blocking agent duration. In a particular embodiment, the durvalumab or effector-silent variant of durvalumab is administered intravenously to an individual at an initial dose of 10 mg/kg over 60 minutes followed by administration of the CTLA-4 blocking agent over 30 minutes on the same day every two weeks as needed.

While the currently approved CTLA-4 blocking agents and PD-1 blocking agents are provided in formulations at a concentration that permits intravenous administration to an individual over a 30 to 60 minute time frame, the combination therapies of the present invention contemplate embodiments in which the CTLA-4 blocking agent and/or the PD-1 blocking agent are each provided in a formulation at a concentration that permits each to be separately administered to an individual in a single injection. Being able to provide at least one of the two blocking agents in a single injection would significantly reduce the time for administering both blocking agent to the individual.

In a further embodiment, the present invention provides a combination therapy in which the CTL-4 blocking agent and the PD-1 blocking agent are co-administered at the same time. Co-administration may be accomplished by providing the CTLA-4 and PD-1 blocking agents in separate formulations and simultaneously providing each formulation to the individual, either by separate IVs or mixing prior to administering the mixture by IV to the individual by IV, or by separate injection of each formulation into the individual. Co-administration may also be accomplished by providing the CTLA-4 and PD-1 blocking agents in a single formulation that is then administered to the individual in a single IV or in a single injection. (i) Combination therapy treatments

The combination therapy of the present invention may be used for the treatment any proliferative disease, in particular, treatment of cancer. In particular embodiments, the combination therapy of the present invention may be used to treat melanoma, non-small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular cancer, non-Hodgkin lymphoma, renal cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, or salivary cancer.

In another embodiment, the combination therapy of the present invention may be used to treat pancreatic cancer, bronchus cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of hematological tissues.

The currently marketed PD-1 blocking agents are approved by the U.S. FDA to treat at least one or more cancers selected from melanoma (metastatic or unresectable), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, MSIHC, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (including advanced), and cutaneous squamous carcinoma. Thus, the combination therapy of the present invention may be used to treat at least one or more cancers selected from melanoma (metastatic or unresectable), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, MSIHC, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (including advanced), and cutaneous squamous carcinoma.

(j) Combination therapy in combination with Chemotherapy

The combination therapy of the present invention may be administered to an individual having a cancer in combination with chemotherapy. The individual may undergo the chemotherapy at the same time the individual is undergoing the combination therapy of the present invention. The individual may undergo the combination therapy of the present invention after the individual has completed chemotherapy. The individual may be administered the chemotherapy after completion of the combination therapy. The combination therapy of the present invention may also be administered to an individual having recurrent or metastatic cancer with disease progression or relapse cancer and who is undergoing chemotherapy or who has completed chemotherapy.

The chemotherapy may include a chemotherapy agent selected from the group consisting of (i) alkylating agents, including but not limited to, bifunctional alkylators, cyclophosphamide, mechlorethamine, chlorambucil, and melphalan;

(ii) monofunctional alkylators, including but not limited to, dacarbazine, nitrosoureas, and temozolomide (oral dacarbazine);

(iii) anthracy dines, including but not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin;

(iv) cytoskeletal disruptors (taxanes), including but not limited to, pacbtaxel, docetaxel, abraxane, and taxotere;

(v) epothilones, including but not limited to, ixabepilone, and utidelone;

(vi) histone deacetylase inhibitors, including but not limited to, vorinostat, and romidepsin;

(vii) inhibitors of topoisomerase i, including but not limited to, irinotecan, and topotecan;

(viii) inhibitors of topoisomerase ii, including but not limited to, etoposide, teniposide, and tafluposide;

(ix) kinase inhibitors, including but not limited to, bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, and vismodegib;

(x) nucleotide analogs and precursor analogs, including but not limited to, azacitidine, azathioprine, fluoropyrimi dines (e.g., such as capecitabine, carmofur, doxifluridine, fluorouracil, and tegafur) cytarabine, , gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and tioguanine (formerly thioguanine);

(xi) peptide antibiotics, including but not limited to, bleomycin and actinomycin; a platinum-based agent, including but not limited to, carboplatin, cisplatin, and oxabplatin;

(xii) retinoids, including but not limited to, tretinoin, abtretinoin, and bexarotene; and (xiii) vinca alkaloids and derivatives, including but not limited to, vinblastine, vincristine, vindesine, and vinorelbine.

Selecting a dose of the chemotherapy agent for chemotherapy depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissue or organ in the individual being treated. The dose of the additional therapeutic agent should be an amount that provides an acceptable level of side effects. Accordingly, the dose amount and dosing frequency of each additional therapeutic agent will depend in part on the particular therapeutic agent, the severity of the cancer being treated, and patient characteristics. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available. See, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis , Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341 : 1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792;

Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343: 1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed); Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002). Determination of the appropriate dose regimen may be made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment, and will depend, for example, the individual's clinical history (e.g., previous therapy), the type and stage of the cancer to be treated and biomarkers of response to one or more of the therapeutic agents in the combination therapy.

For example, pembrolizumab is currently approved by the U.S. FDA for a combination therapy for (i) treating non-small cell lung cancer (NSCLC) comprising

pembrolizumab with pemetrexed and platinum chemotherapy or carboplatin and either paclitaxel or nab-paclitaxel; and (ii) treating head and neck squamous cell cancer (HNSCC) comprising pembrolizumab and platinum-containing chemotherapy, and atezolizumab is currently approved for a combination therapy for treating NSCLC comprising bevacizumab (anti-VEGF-A antibody marketed under the tradename AVASTIN), paclitaxel, and carboplatin.

Thus, the present invention contemplates embodiments of the combination therapy of the present invention that further includes a chemotherapy step comprising platinum- containing chemotherapy, pemetrexed and platinum chemotherapy or carboplatin and either paclitaxel or nab-paclitaxel. In particular embodiments, the combination therapy with a chemotherapy step may be used for treating at least NSCLC and HNSCC.

The combination therapy further in combination with a chemotherapy step may be used for the treatment any proliferative disease, in particular, treatment of cancer. In particular embodiments, the combination therapy of the present invention may be used to treat melanoma, non-small cell lung cancer, head and neck cancer, urothelial cancer, breast cancer,

gastrointestinal cancer, multiple myeloma, hepatocellular cancer, non-Hodgkin lymphoma, renal cancer, Hodgkin lymphoma, mesothelioma, ovarian cancer, small cell lung cancer, esophageal cancer, anal cancer, biliary tract cancer, colorectal cancer, cervical cancer, thyroid cancer, or salivary cancer.

In another embodiment, the combination therapy further in combination with a chemotherapy step may be used to treat pancreatic cancer, bronchus cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of hematological tissues. In particular embodiments, the combination therapy with a chemotherapy step may be used to treat one or more cancers selected from melanoma (metastatic or unresectable), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, MSIHC, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (including advanced), and cutaneous squamous carcinoma.

The following examples are intended to promote a further understanding of the present invention.

EXAMPLE 1

Fc-function is required for induction of gut inflammation irAEs

Gut inflammatory irAEs have been observed in cancer patients during immunotherapy with either anti-CTLA-4 antibody monotherapy or in combination with an anti- PD-1 antibody. Preclinical studies of CTLA-4 deficient mice in constitutive and conditional knock out models have demonstrated development of profound immune mediated inflammatory disease in multiple organs. However, treatment of syngeneic tumor models with surrogate anti- CTLA-4 antibodies has not been reported to induce overt irAEs predictive of the toxicities observed in cancer patients. Similarly, histopathological assessment for gut inflammation was assessed in the CT26 syngeneic or xenograph model (mice inoculated with the CT26 colon carcinoma cell line). CT26 tumor bearing mice treated with Fc-competent anti-mouse CTLA-4 mAh 9D9-mIgG2 _a (a-CTLA-4 or a-CTLA4) (q4x4), resulted in a minimal granulocytic infiltrate

(grade 1 of 5) observed in the lamina propria gut tissues. No granulocytic infiltrates were observed in cohorts treated with Fc-mutant anti-mCTLA-4 mAh 9D9-mIgG _| -D265 A (a-CTLA-

4 (D265A)), Fc-less anti-mCTLA-4 ISVD F894 (CTLA-4 Nab), or isotypes. However, no accompanying ulceration or other tissue damage was observed.

The a-CTLA-4 (D265A) Fc-mutant lacks measurable affinity for Fey receptors (Nimmerjahn et al, Immunity, 23: 41-51 (2005)) and therefor lacks Fc-effector function. In mice, the anti-tumor efficacy of anti-mouse CTLA4 mAh monotherapy is dependent on the ability of the antibody to mediate intra-tumoral regulatory T cell depletion via Fc-effector function (Selby et al, op. cit). As such, both CTLA-4 Nab and a-CTLA-4 (D265A) were not expected to have monotherapy anti-tumor benefit.

The minimal histopathological findings prompted us to evaluate gene expression profiles as potentially more sensitive means to detect markers of inflammatory cell activation in the gut. We utilized a PCR gene-expression panel that we had previously developed for proteomic and expression profiling of genes associated with gut inflammation in fecal samples and biopsies of inflammatory bowel disease (IBD) preclinical models and patients (Cayatte, C, et. al, Clinical and Translational Gastroenterology, 3: elO (2012)). Gene expression profiles were measured in small intestine and colon tissue samples at various times following initiation of treatment with a-CTLA-4 and compared with a- CTLA-4 (D265A) to specifically assess the role of Fc-function for induction gut inflammation. As illustrated in Fig. 1A, expression of numerous gut inflammatory genes was upregulated in the proximal small intestine samples from mice treated with a-CTLA-4 but not from mice treated with a-CTLA-4 (D265A). The results showed that the manifestation of gut inflammatory pathways may be detected by gene expression in small intestine and colon tissue in a subclinical setting.

Upregulation of genes associated with gut inflammation allowed us to assess the effect of sustained treatment on progression to clinical enterocolitis, as observed in ipilimumab treated patients following six to seven weeks or more of treatment (Samaan et al, Nat. Rev. Gastroenterol. Hepatol. 15: 222-234 (2018)). To assess the relative effects of CTLA-4 blockade and Fc-function on gut inflammation and progression to enterocolitis, BALB/c mice were dosed twice weekly with a-CTLA-4, a-CTLA-4 (D265A). Two groups of mice were treated with Fc- competent a-CTLA-4, one group of mice with CT26 tumors and one group of naive BALB/c mice with no tumors, to assess potential contributions from tumor growth and induction of tumor immunity on induction of gut inflammation. Body weights and body condition scores were evaluated twice weekly throughout the sustained treatment to monitor for progression to enterocolitis.

Body weights continued to increase through day 50 in mice dosed with isotype antibodies and a-CTLA-4 (D265A). In contrast, mice dosed with a-CTLA-4 showed a decrease in mean body weight after about 35 to 40 days (Fig. IB). Intestinal permeability was increased in both with a-CTLA-4 treated groups but not in mice treated with a-CTLA-4 (D265A) treated mice, as assessed in FITC-dextran gavaged mice at day 49 and 50 (Fig. 1C). Histologic evidence of inflammation in proximal small intestine and colon as assessed by a pathologist (L.A) revealed progression to moderate and severe enterocolitis in a-CTLA-4 treated groups (Fig. 1 D and Fig. IE), with extensive immune infiltration, thickening of mucosa and loss of goblet cells. In contrast, no enterocolitis was observed in a-CTLA-4 (D265A) treated mice providing evidence that Fc-function is required for CTLA-4 blockade induced enterocolitis. Notably, tumor growth and anti-tumor responses were not required for a-CTLA-4 induced gut inflammation.

It has previously been reported that anti-CTLA-4 antibodies with strong FcyR function are required for strong monotherapy anti-tumor responses in experiments comparing mouse IgG2 _a -chimeric antibodies with high FcyR-affmity with mutant-IgG _| chimeras with no detectable FcyR-binding (Selby et al., Cancer Immunol. Res. 1 : 32-42 (2013)). The specific depletion of T _re g _S in the tumor ostensibly plays a key role in the strong monotherapy observed with anti-CTLA-4 antibodies on the mouse IgG2 _a (mIgG2 _a ) backbone (Simpson et al., J. Exp. Med. 210: 1695-710 (2013). Recent reports demonstrating the importance of CD28 for anti-PD-1 efficacy (See references) suggested to us that augmentation of CD28 function by blocking the stronger interaction of CTLA-4 for ligands CD80 and CD86 with a CTLA-4 antagonist may provide strong combination anti-tumor efficacy without the requirement for T _re g TIL depletion.

ISVDs, specific for mouse CTLA-4 (mCTLA-4), capable of inhibiting binding to CD80 and CD86 were developed using a single-domain VHH antibody fragment derived from heavy chain only camelid antibodies. These small, 15 kDa proteins lack Fc regions and thus do not bind FcyRs. For comparison with a fully Fc-functional antibody, we utilized a-CTLA-4, previously shown to have strong monotherapy anti-tumor efficacy (See references). To control for the potential effects of binding a different CTLA-4 epitope, we utilized a-CTLA-4 (D265 A), which lacks detectable affinity for FcyRs to control for the requirement of strong FcyR-function for anti-tumor efficacy and tolerability.

The CTLA-4 nAb composed of two anti-CTLA-4 VHH domains linked by a 35GS-linker and an anti-human albumin VHH domain as a half-life extension (HLE) subunit See SEQ ID NO:61). The anti-CTLA-4 VHH scored a 96% inhibition for both CD80 and CD86 compared with a-CTLA-4, which was used as a reference antagonist (100%) (Fig. 2A and Fig. 2B). The CTLA-4 nAb was further compared to a-CTLA-4 and effector-silent a-CTLA-4 (D265A) in an in vitro MLR-based bioassay for the capacity to increase proliferation (Fig. 2C), IFNy (Fig. 2D), and IL-2 responses (Fig. 2E).

Fc-function for CTLA-4 blockade is not required in PD-1 combination immunotherapy

The relative anti-tumor efficacy induced by monotherapy with the three CTLA-4 antagonists and in combination therapy with anti-mouse PD-1 antibody mDX400 (a-PD-1) was assessed by measure the change in tumor volume over time in the syngeneic CT26 colon carcinoma tumor model (Fig. 3A-3E), which is only moderately responsive to anti-PD-1 monotherapy. No anti-tumor activity was observed in monotherapy cohorts treated with either CTLA-4 nAb or a-CTLA-4 (D265A) and was comparable to the isotype controls (Group 1 in Fig. 3A). Strong anti-tumor monotherapy activity was observed in mice treated with a-CTLA-4 consistent with prior reports demonstrating the requirement for Fc-function in anti-CTLA-4 antibodies for anti-tumor monotherapy efficacy in mouse syngeneic tumor models (Simpson et al, J. Exp. Med. 210: 1695-710 (2013)) (Group 2 in Fig.3A) and treatment with a-PD-1 alone provided low to modest anti-tumor growth inhibition compared to a-CTLA-4. However, combination therapy with CTLA-4 nAb or a-CTLA-4 (D265A) with a-PD-1 provided strong anti-tumor benefit comparable to that observed with a-CTLA-4 alone or a-CTLA-4 in combination with a-PD-1 (Group 2 of Fig. 3A). The CTLA-4 nAb and a-CTLA-4 (D265A) bind separate epitopes on CTLA-4, thus the effect is not specific to the epitope. Further evidence for robust combination benefit, independent of Fc-function was evident as similar anti- tumor responses were observed in a-PD-1 combination treatments between CTLA-4 nAb, a- CTLA-4 (D265A), and a-CTLA-4. Fig. 3D shows the individual results for each of the 10 mice treatments summarized in Fig. 3A. Expansion of CD8 T cells and increased CD8/Treg ratios were observed in a-CTLA-4 treated mice and in mice receiving a combination of a-CTLA-4 (D265S) plus a-PD-1 treated mice compared to mice that received a-CTLA-4 (D265S) alone (Fig. 3B). These results indicate that anti-CTLA-4 antagonists, lacking in Fc-function, combined with an anti -PD- 1 antagonist provided superior anti -tumor efficacy than that achievable with anti-PD-1 antagonist monotherapy and provided anti -tumor efficacy in the combination similar in effect as the anti -tumor efficacy of anti-CTLA-4 antibodies with Fc- function in a monotherapy.

We investigated regulation of immune response genes associated with effective cancer immunotherapy in tumors on nine-days after initiation of treatment to elucidate potential complimentary mechanisms associated with the strong combination activity. PCR-expression profiling of tumors from mice treated with a-CTLA-4 showed strong upregulation of numerous genes associated with effective immunotherapy (Fig. 3C), including IFNy. IFN-response genes, chemokines, pro-inflammatory cytokines, and MHC. Only modest upregulation was observed in tumors from a-CTLA-4 (D265A) treated mice indicating that the strong upregulation observed in a-CTLA-4 treated tumors was at least partially dependent on Fc-function. Modest responses were also observed in tumors from CTLA-4 nAb and a-PD-1 treated mice. In contrast, robust upregulation of tumor immune response genes was observed in combination therapy cohorts treated with CTLA-4 nAb or a-CTLA-4 (D265A) plus a-PD-1. Fig. 3D shows neither CTLA-4 nAb or a-CTLA-4 (D265A) has anti -tumor activity in the absence of the PD-1 blockade. These data support the hypothesis that complimentary mechanisms for pure CTLA-4 blockade and PD- 1 blockade can provide strong combination benefit in an Fc-independent manner.

CTLA-4 blockade without Fc-function combined with anti-PD-1 provides superior therapeutic index

A prominent feature of immune checkpoint blockade is clinically validated combination benefit of anti-PD-1 and anti-CTLA-4 antibodies resulting in superior clinical efficacy when compared to targeting either checkpoint pathway alone. However, immune- related toxi cities (irAEs) associated with CTLA-4 blockade combination therapy with anti-PD-1 have been associated with increased induction of gut inflammation in patients (Ribas &

Wolchok, Science 359: 1350-1355 (2018). In addition, both a-CTLA (D265A) and Fc-less CTLA-4 nAb required combination with a-PD-1 in order to induce strong anti-tumor immunity. To control for potential effects of strong tumor immunity on induction of gut inflammation, we examined gut inflammation expression profiles in mice receiving combination therapy with a- PD-1 plus either a-CTLA, a-CTLA (D265A), or CTLA-4 nAb after five-treatments, on day 18 after initiation of treatment (Fig. 4D).

To assess the relative effects of CTLA-4 blockade and Fc-function on gut inflammation, naive BALB/c mice were dosed twice weekly with a-CTLA, a-CTLA (D265A), Fc-less CTLA4 nAb, a-PD-1, or with combinations of a-PD-1 with the various anti-mCTLA4 antagonists. Body weights and body condition scores were evaluated twice weekly throughout the study. Mouse body weights in all groups increased through approximately Day 20 (Fig. 4A). Body weights continued to increase through day 50 in mice dosed with isotype, a-CTLA

(D265A), CTLA4 nAb, or a-PD-1 or combinations thereof. Mice dosed with a-CTLA showed a decrease in mean body weight after about day 30 to near pre-treatment levels by day 50.

Administration of a-CTLA in combination with a-PD-1 led to a more rapid decrease in mean body weight below pre-treatment levels from day 20 through the day 50. Notably, mice dosed with a-CTLA, the body condition score for 2 of 8 mice dropped to 2 (under-conditioned) beginning on day 42 and beginning on day 28 in mice treated with a-CTLA in combination with a-PD-1. These cohorts presented glossy, scruffy fur, and swollen abdomen were observed in these cohorts.

Analysis of inflammation was scheduled after seven weeks of dosing, when mice dosed with a-CTLA showed loss of body weight over the time period, which was exacerbated when administered in combination with a-PD-1 (Group B in Fig. 4A). In contrast, none of the effector-silent CTLA-4 blocking agents or a-PD-1 showed any significant weight loss compared to the isotype controls during the time period (Group A in Fig. 4B).

All mice from the combination treatment groups and four mice from the isotype control and a-PD-1 treatment groups were euthanized on day 50 for tissue collection. The four remaining mice from the isotype control and a-PD-1 treatment groups and all mice from the single agent treatment groups were euthanized on day 54 for tissue collection. At the time of necropsy, the proximal small intestine and colon were resected for RT-qPCR to determine the expression of the inflammatory genes and for assessment of inflammation by histopathology.

A heat map of gene expression in the proximal small intestine from each treatment group relative to isotype control is shown in (Fig. 4D). Administration of a-CTLA was sufficient to induce upregulation of inflammatory genes in the jejunum (Fig. 4D) and colon (Fig. 4E). The combination of a-CTLA with a-PD-1 induced an even stronger upregulation of inflammatory genes than the a-CTLA monotherapy. In contrast, administration of CTLA-4 nAb induced little or no gut inflammatory gene expression and only modest upregulation when combined with a-PD-1. Similarly, administration of a-CTLA (D265A) alone or in combination with a-PD-1 resulted in minimal to low induction of inflammatory genes. Gut permeability, assessed in serum after FITC-dextran gavage, was significantly increased in mice treated with a- CTLA and mice receiving combination treatment with a-CTLA and a-PD-1. Severity of inflammation in the proximal small intestine was scored by histological assessment of enteritis in proximal jejunum on day 50. By histopathological assessment, administration of a-CTLA resulted in mild to severe inflammation in most mice. In cohorts treated with a combination of a-CTLA and a-PD-1, sustained treatment induced moderate to very severe inflammation in all mice (Fig. 4B). Mice with very severe enteritis, presented with jejunitis, diffuse neutrophilic lesions with moderate numbers of mast cells and degeneration of neurons of Meissner plexus. In contrast, administration of either CTLA-4 nAb or a-CTLA (D265A) did not induce inflammation in the histopathology assessment. Administration of CTLA-4 nAb in combination with a-PD-1 led to no inflammation or minimal to mild inflammation in several mice. Administration of a-CTLA (D265A) in combination with a-PD-1, led to mild inflammation in only one of the eight mice. Representative photomicrographs demonstrate the relative level of inflammation in each treatment group (Fig. 4C).

As shown in Figs. 5A-5C, a-CTLA-4 Fc effector function drives skin inflammation (Fig. 5A) but not systemic inflammation, where there was no detectable inflammation in kidney, liver, or lung (Fig. 5C). Absolute number of ear skin IL-17-producing T cells, Foxp3+ Treg cells and neutrophils were measured by flow cytometry. As shown in Fig. 5B, elevated levels of IL-17-producing T cells, Foxp3+ Treg cells and neutrophils were present in ear skin from mice treated with a-CTLA-4 but not with a-CTLA-4 (D265A). Together, these data support a key role of the Fc-effector function in the induction of gut inflammation by anti mouse CTLA-4 antibodies having effector function. Fc-effector function contributed to anti mouse a-CTLA induced gut inflammation in the B ALB/c mouse model of enterocolitis, whereas gut inflammation is mild or absent in mice treated with CTLA-4 nAb or in mice treated with a- CTLA (D265A).

In summary, two attributes were associated with induction of gut inflammation in the CT26 tumor model by a-CTLA-4. First, CTLA-4 specificity was required as Fc-functional isotype controls did not elicit gene expression associated with inflammation. However, blocking of CTLA-4 binding to CD80/CD86 ligands was insufficient to induce upregulation of inflammatory genes in the bowels of the CTLA-4 nAb and the a-CTLA-4 (D265A) treated mice. Hence, the strong Fc-function capacity present in the IgG2 _a isoform in the a-CTLA-4 was required for induction of gut inflammation.

Activation of gut inflammation is initiated by activation of T _e cells independent of depletion of

7j regs

The mechanism of action (MO A) for anti-CTLA-4 mediated anti-tumor immunity is theoretically mediated by pharmacodynamics (PD) effects on T regulatory (T _re g) cells as well as on T effector (T _e ff) cell populations (CTL, TH1 cells, etc.). Depletion of T _re g cells within the tumor microenvironment (TILs) is a prominent MOA for anti-CTLA-4 antibodies in murine syngeneic tumor models (Simpson et al, op. cit.). Additionally, Fc-FcyR co-engagement by anti- CTLA-4 mAbs modulates T cell receptor (TCR) and CD28 signaling resulting in enhanced T cell activation independent of T _re g depletion (Waight et al., Cancer Cell, 33: 1033-1047 (2018)).

To characterize differential effects of effects of a-CTLA-4 from CTLA-4 nAb on T _re gs ^ar| d T effector cells, flow cytometry was conducted on T cell populations from tumor

(TILs), lamina propria of the colon, blood and spleen 20-hours following subcutaneous administration. We were able to measure CTLA-4 expression levels in T _re g _S from mice treated with a-CTLA-4 or a-CTLA-4 (D265A) using anti-CTLA-4 mAh clone UC10-4B9

(ThermoFisher) as they do not cross-block, enabling staining of drug-bound CTLA-4. As reported previously in the literature (Selby et al. op. cit, Simpson et al. op. cit), we observed differential expression of CTLA-4 in T _re g _S within spleen (CTLA-4^ ⁰ ) and tumor

microenvironments (T _re „hi) of CT26 tumor bearing mice. T _re g _S within PBMC expressed bi- modal levels of CTLA-d' ^{0 1} ™^ Interestingly, T _re g _S from the lamina propria of the colon expressed bi-modal levels of CTLAA^d-hi xh _e CTLA-d^i T _re g _S in the colon expressed similar levels to Treg TIL populations. The differential expression levels of CTLA-4 on the various T cell populations impacts the capacity for ADCC mediated depletion due to receptor density dependent killing mechanisms. While T _re g populations normally express higher levels of CTLA-4, T _re g _S in the tumor environment express much higher levels (3.3 -fold higher, MFI =

8,100 in isotype controls of Treg TILs) than those found in spleen (MFI= 2,400). Lamina propria T _re g _S from the colon which expressed higher CTLA-4 levels (CTLA-4hi mode MFI = 10,000) resembled T _re g TILs for relative expression levels using flow cytometry (Fig. 6A). As illustrated in Figs. 6B- 6D, significant depletion of T _re g _S was limited to TILs from the tumor microenvironment of mice treated with a-CTLA-4, which have the highest density of CTLA-4 expression. Treatment with Fc-mutant a-CTLA-4 (D265A), which lacks Fc-function, did not result in T _re g depletion.

Based merely on the assumption that cells expressing higher CTLA-4 levels would be predisposed for depletion by a-CTLA, we predicted that CTLA-4hi T _re g _S in the Lamina propria would be depleted, similar to T _re g TIL populations. Surprisingly, only T _re g _S

TILs isolated from tumors of a-CTLA-4 treated mice appeared to be depleted. Lamina propria derived T _re g _S from colons of a-CTLA-4 treated mice did not appear to be depleted. No detectable depletion of lamina propria derived T _re g _S from colons of a-CTLA-4 treated mice was observed, suggesting that that the induction of gut inflammation was not initiated by a loss of Tregs i ⁿ the gut mucosa.

We investigated possible phenotypic changes in T _re g _S effecting suppressor function that may have contributed to a-CTLA-4 induced gut inflammation. Colonic lamina propria (LP) T _re g _S from MC38 tumor implanted FoxP3 GLD reporter mice were sorted for PCR expression profiling of genes associated with T _re g function (Fig. 7A). However, no significant gene expression differences were observed in LP T _re g cells from a-CTLA compared to a- CTLA-4 (D265A) treated mice. The potential effect of Fc-function on T _re g _S was further investigated using the CD45RBhi T cell transfer model of colitis. Passive transfer of T _re g cells with CD45RB^i T cells protected mice from development of Colitis (Fig. 7B-7C). Treatment of mice co-administered T _re g cells with CD45RBhi T cells with a-CTLA-4 resulted in a loss of T _re g protection and development of colitis. In contrast, loss of protection was not observed in

CTLA-4 nAb treated mice (Fig. 7B-7C). Gene expression profile from flow cytometry sorted colon Foxp3+ T _re g cells from mice 24 hours post treatment showed significant upregulation of gene expression from mice treated with a-CTLA-4, which is similar to that observed with CD45RBhigh, compared to the gene expression observed with cells obtained from mice treated with CTLA-4 nAb or isotype controls. Collectively, these results suggest Fc-mediated T _re g depletion is not essential for induction of gut inflammation by a-CTLA-4 but the regulatory function of T _re g _S in response to gut inflammation may be modulated.

The restricted expression of CTLA-4 and CD28 on T cells and CD80, CD86 and

FcyR on antigen presenting cells may play a key role for Fc-functional a-CTLA-4 nAb activation of T cells independent of T _re g depletion (Waight et. al., Cancer Cell, 33: 1033-1047, 2018). Fc- enhanced activation in tumors is advantageous but could contribute to inadvertent irAEs in gut tissues. We investigated potential contribution of Fc-function in immune effector cell activation in gut tissues. Flow cytometric analysis of CD 16/32 expression on macrophages from both tumor and colon showed significantly higher level of FcR on antigen presenting cells compared to splenic macrophages (Fig. 8A & 8B). Additionally, the proportion of CD45 ⁺ CD1 lb ⁺ F4/80 ⁺ macrophages in tumor and colon lamina propria was substantially higher than in spleen (Fig.

8C). Fc-function was required for activation o G I L 1 b . TNFa and IFNy cytokine responses in gut tissues and were evident as early as 10-days after initiation of treatment, well before overt evidence of gut inflammation suggesting a key role in irAE induction (Fig. 8C).

Cytokine responses in CD4 T cells isolated from colon lamina propria of a- CTLA-4 treated mice after one month of treatment showed a higher proportion of IL-17, TNFa and IFNy producing cells associated with Fc-mediated gut inflammation (Fig. 8E). Additionally, an Fc-dependent increase in neutrophils was also induced in a-CTLA-4 treated mice (Fig. 8E). Collectively, the results indicate CTLA-4 blockade associated gut inflammation in induced by Fc-mediated activation of effector cells, augmented by FcyR-antibody enhancement of cellular bridging of APC with T cells resulting in stimulation of inflammatory cytokine responses as illustrated in Fig. 11. Fc-mediated induction of gut inflammation can be induced by Effector T cells, independent of Treg depletion.

Discussion Several recent reports have supported a key role for Fc-function for CTLA-4 blockade monotherapy in syngeneic mouse cancer immunotherapy tumor models (International patent Application WO 2014/089113; Selby et al, Cancer Immunol. Res. 32-42 (2013); Vargas et al, Cancer Cell, 33: 649-663 (2018)). Ingram et al. reported in Proc. Natl. Acad. Sci. USA 115: 3912-3917, (2018) that to provide an anti-CTLA-4 ISVD with anti-tumor efficacy required fusing an Fc domain to the ISVD. Indeed, we observed similar lack of efficacy when treating tumors with CTLA-4 nAb or a-CTLA-4 (D265A) as a monotherapy. Specific depletion of tumor infiltrating T _re g _S , which express higher cell surface CTLA-4 levels, has been

demonstrated to contribute to tumor efficacy in tumor models (Simpson et al., J. Exp. Med., 210: 1695-1710 (2013); Selby et al, Cancer Immunol. Res 1 : 32-42 (2013).

The induction of enterocolitis by a-CTLA-4 was not associated with detectable depletion of T _re g in residing in the lamina propria. Our work expands on the potential for

CTLA-4 blockade in anti-cancer treatments by demonstrating strong anti-tumor activity when combined with anti-PD-1 antibodies and without the induction strong gut inflammation. Fc- function was not required to achieve combination benefit for the CTLA-4 and PD-1 blockades. Combination treatments comprising a CTLA-4 nAb or a-CTLA-4 (D265A) with a-PD-1 induced similar activation of IFNy-associated immune response genes in tumors as those induced by a- CTLA-4 bearing strong Fc-function. In contrast, strong gut inflammation progressing to enterocolitis was primarily observed in mice treated with a-CTLA-4 and was increased when combined with anti-PD-1 antibody mDX400. These results indicate that simple blockade of CTLA-4, facilitating activation of CD28, is sufficient to increase anti-tumor responses of exhausted T cells when combined with PD-1 blockade.

A previous report by Kamphorst et al. in Science 355: 1423-1427 (2017) demonstrated that PD-1 blockade rescue of exhausted CD8 T cells requires CD28 co-stimulation of TCR activation. Moreover, a companion report by Hui et.al. in Science 355: 1428-1433 (2017) demonstrated that the co-receptor, CD28, is strongly preferred over the TCR as a target for dephosphorylation by PD-1 -recruited Shp2 phosphatase and that CD28 is preferentially dephosphorylated. Our results show that the complimentary activation of the TCR co-receptor CD28 mediated by simple blocking of CTLA-4 by anti-CTLA-4 antibodies without Fc-function and blockade of PD-1 mediated dephosphorylation of CD28 may be sufficient to achieve a combination benefit for cancer immunotherapy. The advantage of a simple combination blockade, without Fc-mediated enhanced activation through Fc-FcyR bridging, may be it permits a larger therapeutic index that enables a higher dose range and longer treatment times. This advantage may also facilitate further combinations with chemotherapeutic standards of care due to a lower gut inflammation irAE risk profile.

EXPERIMENTAL PROCEDURES Mice

Wild-type C57BL/6J mice were obtained from Jackson laboratories. Wild-type Balb/c and CB17-SCID mice were obtained from Taconic. B6.Foxp3GDL (GFP-DTR- luciferase) mice generated and maintained under specific pathogen-free conditions and kept in microisolators with filtered air at the Merck Research Laboratories (MRL) animal facility at Palo Alto, California. All animal procedures were approved by the Institutional Animal Care and Use Committee of MRL in accordance with guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care.

Tumor challenge and treatment

For syngeneic tumor experiments, the CT26, MC38, and MB49 tumor models were used. 8- to 12-week-old Balb/c or C57BL/6J mice were subcutaneously (s.c.) injected with 3 x 1 (P CT26 cells on the flank. Tumor diameter was measured by electronic calipers and tumor volume was calculated by length ^c width ^c width ^c 1/2. Treatments were started when tumors reached approximately 100 mm-L Mice were treated twice a week subcutaneously (s.c.) with a- CTLA-4, a-CTLA-4 (D265A), mouse anti-IgG _| - D265A antibody isotype control, mouse anti-

IgG2 _a antibody isotype control, a-PD-1 antibodies at 10 mg/kg. Mice were treated twice a week s.c. with CTLA-4 nAb or ISVD control at 30mg/kg.

a-CTLA-4 comprises a HC having the amino acid sequence set forth in SEQ ID NO:58 and a LC having the amino acid sequence of SEQ ID NO:59.

a-CTLA-4 (D265 A) comprises a HC having the amino acid sequence set forth in SEQ ID NO:60 and a LC having the amino acid sequence of SEQ ID NO:60.

a-PD-1 comprises a HC having the amino acid sequence set forth in SEQ ID NO:63 and a LC having the amino acid sequence of SEQ ID NO:64.

CTLA-4 nAb comprises the amino acid sequence set forth in SEQ ID NO:61. Anti-PD-1 ISVD F037 (PD-1 nAb) comprises the amino acid sequence set forth in SEQ ID NO:62.

Induction of colitis and skin inflammation

Naive 8- to 12-week-old Balb/c mice were treated twice a week s.c. for eight weeks with a-CTLA-4, a-CTLA-4 (D265A), mouse anti-IgG _| -D265A isotype control, mouse anti-IgG2 _a isotype control, antibodies at 20 mg/kg. Mice were treated twice a week s.c. with CTLA-4 nAb or ISVD control at 30 mg/kg. at day 55, Plasma was collected for ELISA and Luminex assays. Organs were collected and treated as follows: 1) fixed in 10 % neutral buffered formalin and stained paraffin-embedded tissue sections with H&E to evaluate tissue pathology; 2) snap-frozen in liquid nitrogen for further RNA extraction; or 3) placed in HBSS for cell isolation. T cell-driven colitis

Spleen cells from Balb/c mice were processed and purified for CD4 using magnetic bead separation (STEM CELL Technologies). TCRb+ CD4+ CD25- CD45RBhigh T cells (CD45RBhigh T cells) and TCRb+ CD4+ CD25+ CD45RBlow (T _re g cells) were sorted with FACS Aria (BD). 3\ 1 (P CD45RB^igh T cells and 1 x 1 (P T _re g cells were injected intravenously. Mice were dosed i.p. twice a week with 350 pg of a-CTLA-4 or isotype control or 600 pg of CTLA-4 nAb or ISVD control. Mice were monitored and weighed for seven weeks post injection.

Intestinal permeability

Mice were gavaged with FITC-Dextran (4kDa, Sigma-Aldrich) four hours prior to fluorescence measurement of FITC in the serum.

Colon lamina propria, skin and tumor cell isolation

Colon lamina propria cells were isolated by first removing epithelial cells through the incubation of 0.5-cm gut tissue pieces in Hank’s buffered salt solution containing 5 mM EDTA and 10 mM HEPES for 20 minutes at 37°C and then repeating this incubation one additional time. The remaining tissue was cut into small fragments and then digested with HBSS IX medium containing 0.250 mg/mL LIBERASE (Roche), 30 U/mL DNase I (Sigma-Aldrich) and DISPASE (Coming) at the same conditions. The resulting cell suspension was layered on to a 40%/80% PERCOLL gradient and centrifuged for 10 minutes at 600 g; LP cells were recovered at the interface.

Ear skins were chopped and digested HBSS IX medium containing 0.250 mg/mL LIBERASE (Roche), 30 U/mL DNase I (Sigma-Aldrich) and DISPASE (Coming) for 90 minutes at 37°C. Cell suspension was filtered and washed twice with HBSS IX buffer. Tumors were chopped and digested HBSS IX medium containing 0.250 mg/mL LIBERASE (Roche),

30 U/mL DNase I (Sigma-Aldrich) and DISPASE (Coming) for 30 minutes at 37°C. Cell suspension was filtered and washed twice with HBSS IX buffer.

Histology from colon, ear skin, liver, lung, kidney strips were fixed in 10% neutral buffered formalin overnight, transferred to 70% ethanol, processed routinely, embedded in paraffin, sectioned at 4-5 pm, then stained with hematoxylin and eosin (H & E). Colons were scored for severity of disease by a pathologist in a blinded fashion according to three criteria: Inflammation: when present was characterized by infiltration with large numbers (60-70%) of mononuclear cells (macrophages and lymphocytes) and 30-40 % of neutrophils and band cells. The scoring of inflammation includes severity of infiltration, loss of glands, erosion, dilatation of glandular lumina, presence of crypt abscess and degeneration of epithelial cells. Inflammation was scored on a scale of 0-4, 0 = negative; 1 = minimum, 2 = mild; 3 = moderate; 4 severe. Apoptosis: The prevalence of apoptotic bodies were scored on a scale of 0-3, 0 = negative; 1 = low, 2 = moderate; 3 = high. Regeneration: Regenerative changes assessed include scoring of the prevalence of mitotic figures in the upper 1/3 of the mucosa, nuclear density (nuclear crowding) within individual glandular structures, regularity of the surface epithelium. Apoptosis was scored on a scale of 0-3, 0 = negative; 1 = low, 2 = moderate; 3 = high.

Flow cytometry and antibodies

Cells were resuspended in PBS and stained on ice for 30 minutes in the dark with a fixable viability stain (BD Bioscience). Then, cells were resuspended into the stain buffer (FBS, BD bioscience) and stained on ice for 30 minutes with various combinations of directly fluorochrome-conjugated. For intracellular antigens, surface stained cells were permeabilized, fixed with Foxp3 staining buffer set (eBiosciences) for 30 minutes on ice and then stained with specific antibodies. Mouse antibodies: CD45 (30-F11), CD8a (53-6.7), CTLA-4 (UC10-4B9), CDl lc (HL3), CDl lb (Ml/70), TCR (H57-597), TCRyd (GL3), CD4 (RM4-5 or GK1.5),

CD25 (PC61), CD45RB (16A), Ly6G (1A8), F4/80 (T45-2342), CD16/32 (2.4G2), IFNy (XMG1.2), IL-17A (TCI 1-18H10), TNFa (MP6-XT22), Foxp3 (FJK-16s). All of the antibodies were purchased from BD biosciences, Biolegend or eBioscience. For all samples, acquisition was performed on LSR II flow cytometer (BD). Data were analyzed using FLOWJO software (Tree Star).

When cytokine production was measured by flow cytometry, cells were stimulated with 500 ng/mL Ionomycin, 50 ng/mL PMA (Sigma-Aldrich). After one hour, Brefeldin A (BD Bioscience) was added for another two hours prior to staining.

Mouse allogeneic mixed leukocyte reaction (MI.R) assay

2.105 Mouse C57B6/J (8-12 weeks old, female) splenic T cells were isolated using the EASYSEP Mouse T Cell Isolation Kit (STEMCELL) and co-culture with 1 x 10^ irradiated (at 2000 rad) Balb/c mouse splenocytes in the presence of indicated concentration of a-CTLA-4, a-CTLA-4 (D265A), CTLA-4 nAb or isotypes controls. At day three, supernatant was collected and IL-2 and IFN-gamma production were measured by ELISA according to manufacturer’s protocol (Meso Scale Discovery). Cells were then pulsed with [¾] -thymidine (1 pCi per well) for six hours or 16-18 hours. Cells were harvested onto glass fiber filters using a cell harvester. Filters were counted in a MicroBeta plate counter (PerkinElmer Microbeta 2450) according to manufacturer’s instruction.

Total RNA isolation from tissues and cells and subsequent gene expression analysis using the Fluidigm BIOMARK platform. For real-time PCR analysis, total RNA was isolated by either of two methods. Organs were homogenized in RNA STAT-60 (Tel-Test Inc., Friendswood, TX) with a polytron homogenizer and then RNA extraction was performed with the MagMAX-96 for Microarrays Kit (ThermoFisher Scientific, Waltham, MA) per manufacturer's instructions. For cellular samples, RNA was isolated using the ARCTURUS PICOPURE RNA Isolation Kit per manufacturer’s instructions (ThermoFisher Scientific, Waltham, MA).

DNase-treated total RNA was reverse-transcribed using QUANTITECT Reverse Transcription (Qiagen, Valencia, CA) per manufacturer's instructions. Primers were obtained commercially from ThermoFisher Scientific (Foster City, CA). Gene specific pre-amplification was done on at least 2 ng cDNA per Fluidigm BIOMARK manufacturer's instructions (Fluidigm, Foster City). Real-time quantitative PCR was then done on the Fluidigm BIOMARK using two unlabeled primers at 900 nM each and 250 nM of FAM-labeled probe (ThermoFisher Scientific, Foster City, CA) with TAQMAN Universal PCR Master Mix containing UNG. Samples and primers were run on either a 48x48 array or 96x96 array per manufacturer's instructions

(Fluidigm, Foster City). Ubiquitin levels were measured in a separate reaction and used to normalize the data by the ACt method. (Using the mean cycle threshold value for ubiquitin and the gene of interest for each sample, the equation 1.8 ^L (Ct ubiquitin minus Ct gene of interest) x 104 was used to obtain the normalized values.). Primer references sequences are available on demand.

Statistics

Two-tailed paired and unpaired t test were used to calculate statistical significance in the rest of this study. * PO.05, ** PO.01, *** P<0.001. Statistics were performed using GraphPad PRISM 7 software.

EXAMPLE 2

The anti-tumor efficacy of CTLA-4 nAb was assessed in the mouse syngeneic MB49 tumor model. MB49 cells are a urothelial carcinoma line derived from an adult C57BL/6 mouse by exposure of primary bladder epithelial cell explants to 7, 12-dimethylbenz[a] anthracene (DMBA) for 24 hours followed by long-term culture. The syngeneic murine model of bladder cancer has been widely used for more than 35 years.

MB49 mouse bladder cancer cells were implanted subcutaneously (s.c.) into 80 mice and animals were assigned to five treatment groups with 10 mice each. When the median starting tumor volume reached 103 mm^ mice were injected s.c. once every four days for a total of four doses. An irrelevant control ISVD (30 mg/kg, lot number 01AQL) and 5 mg/kg mlgGi isotype control mAh (lot number 64AIS) were administered as a treatment control. Treatments included 30 mg/kg CTLA-4 nAb, 10 mg/kg Fc-competent a-CTLA-4 (D265A), 5 mg/kg a-PD-1, or combinations of CTLA4 targeting agents and a-PD-1. Tumor growth was monitored for 21 days post treatment initiation.

Fig. 9A shows the individual animal tumor volumes for each treatment group. Complete responses (CR) through Day 21 are presented for responsive treatment groups. Fig. 9B shows the mean tumor volume and standard error of the mean for each treatment group (starting number n=10/group). Tumor volumes form animals that were removed from the study due to large tumor volumes were carried forward in the mean until the last measurement was taken for that treatment group. Figs. 9A-9B show that like in the CT26 colon tumor model (see Fig. 3A), the MB49 bladder tumor model and in the MC38 colon tumor models, combination therapy with Fc-less CTLA-4 nAb with a-PD-1 provided strong anti-tumor benefit, independent of Fc- function.

EXAMPLE 3

The anti-tumor efficacy of CTLA-4 nAb was assessed in the mouse syngeneic MC38 tumor model. MC38 mouse colon cancer cells were implanted SC into 80 mice and animals were assigned to five treatment groups with 10 mice each. When the median starting tumor volume reached 246 mm·’ mice were injected SC once every four days for a total of four doses. An irrelevant control ISVD (30 mg/kg) and 5 mg/kg mlgG^ isotype control mAh were administered as a treatment control. Treatments included 30 mg/kg CTLA-4 nAb, 10 mg/kg Fc- competent a-CTLA-4 (D265A), 5 mg/kg a-PD-1, or combinations of CTLA4 targeting agents and a-PD-1. Tumor growth was monitored for 23 days post treatment initiation.

Fig. 10A shows the individual animal tumor volumes for each treatment group. Complete responses (CR) through Day 23 are presented for responsive treatment groups. Fig. 10B shows the mean tumor volume and standard error of the mean for each treatment group (starting number n=10/group). Tumor volumes form animals that were removed from the study due to large tumor volumes were carried forward in the mean until the last measurement was taken for that treatment group. Figs. 10A-10B show that like in the CT26 colon tumor model (see Fig. 3A), the MB49 bladder tumor model and in the MC38 colon tumor models, combination therapy with Fc-less CTLA-4 nAb with a-PD-1 provided strong anti -tumor benefit, independent of Fc-function.

Sequences

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While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.

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