Dosing regimen of gp100-specific TCR-anti-CD3 scFv fusion protein

Abstract

This disclosure relates to the treatment of cancer, particularly gp100-positive cancers. Specifically, this disclosure relates to dosing regimens for T cell redirecting bispecific therapeutic agents comprising a targeting moiety that binds to the YLEPGPVTA-HLA-A2 complex fused to a CD3-binding, T cell redirecting moiety. [Selection diagram] None

Description

[Technical field]

[0001] The present invention relates to the treatment of cancer, particularly gp100 positive cancers. In particular, the present invention relates to a dosing regimen of a bispecific T cell redirecting therapeutic comprising a targeting moiety that binds the YLEPGPVTA-HLA-A2 complex fused to a CD3 binding T cell redirecting moiety. [Background technology]

[0002] Human glycoprotein 100 (gp100) is one of the melanoma-associated antigens that can elicit a natural immune response in the human body. This protein is a 661 amino acid melanosome membrane-associated glycoprotein that is expressed in normal melanocytes and is widely overexpressed in many melanoma cancer cells. For example, one study (Trefzer et al. (2006), Melanoma Res. 16(2):137-45) found that 82% of 192 melanoma metastases from 28 melanoma patients expressed gp100. Several studies reported higher expression levels of gp100 in melanoma tissues (Hofbauer et al. (2004), J Immunother. 27(1):73-78; Barrow et al. (2006), Clin Cancer Res. 12:764-71). Although the majority of gp100-positive cancers are melanomas, the literature contains numerous reports of other forms of cancer, such as clear cell sarcoma and various brain cancer subtypes, that express gp100, as detected by immunohistochemistry using, for example, an antibody called HMB-45, a widely used gp100-specific diagnostic antibody (Huang et al. (2015), Int J Clin Exp Pathol. 8(2):2171-5; Ozuguz et al. (2014), Indian Dermatol Online J 5(4):488-90; Taddei et al., Appl Immunohistochem Mol Morphol (2001) 9(1):35-41). The exact function of this protein is unknown, but it appears to be involved in melanosome maturation (Hoashi et al., 2005; Kawakami and Rosenberg, 1997). The gp100 antigen has been and continues to be the target of several immunotherapy-based melanoma clinical trials.

[0003] Early gp100-targeted immunotherapy included vaccination strategies using peptides or viruses expressing the entire or partial gp100 protein (reviewed in Lens (2008), Expert Opin Biol Ther. 8(3):315-323). These trials met with limited success, which is likely a result of the generation of an inadequate immune response rather than a reflection of the relevance of the target. Thus, the basis of using gp100 as a melanoma target remains a valid approach for melanoma-specific immunotherapy. The development of novel bispecific therapeutic agents comprising a T cell receptor-based gp100-specific targeting moiety fused to a CD3-binding T cell redirecting moiety provides a new therapeutic option (Liddy et al. (2012), Nat Med. 8:980-987). The mechanism of action of such agents is significantly different from other previous immunotherapies, resulting in rapid and potent redirection of non-gp100-specific T cells to kill gp100-positive cells in vitro; thus, there is reason to expect both improved clinical efficacy in patients and on-target, off-tumour toxicity due to activity against gp100-positive normal tissues (e.g., skin melanocytes).

[0004] The peptide YLEPGPVTA (SEQ ID NO: 1) corresponds to amino acid residues 280 to 288 of gp100. The YLEPGPVTA (SEQ ID NO: 1) peptide is bound to gp100 by class I HLA molecules. + / HLA-A * 02 + Presented on cancer cells (Salgaller et al. (1996), Cancer Res 56:4749-4757). WO2011 / 001152 discloses peptide-HLA-A *We describe a TCR that binds to the YLEPGPVTA peptide presented as a 02 complex. This TCR can be mutated to native gp100 TCR α and / or β variable domains to have improved binding affinity and / or binding half-life for the complex, and linked (covalently or otherwise) to a therapeutic agent. One such therapeutic agent is an anti-CD3 antibody or a functional fragment or variant thereof (e.g., a single chain variable fragment (scFv)). The anti-CD3 antibody or fragment can be covalently linked to the C-terminus or N-terminus of the α or β chain of the TCR. This TCR has been used to treat melanoma in patients (clinical trial identification number NCT01211262).

[0005] IMCgp100 is the YLEPGPVTA peptide-HLA-A * The IMCgp100 is a bispecific therapeutic agent for redirecting T cells, comprising a soluble, affinity-enhanced TCR that binds to the 02 complex, fused to an anti-CD3 scFv. IMCgp100 has been shown to function in vitro by binding to gp100-positive target cells and inducing killing through the recruitment of both tumor-specific and non-tumor-specific T cells. * Upon binding to the 02 complex, IMCgp100 bridges the target cell to a T cell through the interaction of its anti-CD3 scFv portion. Cross-linking of these two cells with a bispecific therapeutic agent results in the formation of an immune synapse, activation of T cells, and redirection of the T cell response to gp100-positive target cells. IMCgp100 binds to CD4 + T cells and CD8 + However, its primary function is to activate both CD8 and T cells. + The aim of this study was to induce T cell responses. + T cells, also known as cytotoxic T lymphocytes (CTLs), play an important role in the innate immune system's repertoire for fighting cancer. CTLs are cytolytic and function to induce the lysis of tumor cells through the apoptotic pathway.

[0006] As mentioned above, gp100 is expressed in normal healthy cells, including melanocytes, which raises the possibility of on-target off-tumor reactivity in clinical situations. It is known that the expression level of gp100 in melanocytes is lower than in tumors. During preclinical testing of IMCgp100, it has been established that IMCgp100 is capable of redirecting T cell activity against normal melanocytes (albeit with lower efficacy than tumor cells; in this regard, efficacy is directly proportional to the number of epitopes displayed on the surface of the target cells) (Liddy et al. (2012), Nat Med. 8:980-987). While these data indicate that there may be a therapeutic window between on-tumor and off-tumor reactivity, on-target off-tumor reactivity cannot be excluded by IMCgp100 treatment. During clinical trials, any reactivity against skin melanocytes was expected to manifest as skin-related toxicity (e.g., rash, vitiligo, and local cytokine and chemokine release effects; these could be addressed with hydrocortisone treatment). No other safety concerns were identified during in vitro preclinical testing of IMCgp100, including no evidence of off-target cross-reactivity. Based on this data, IMCgp100 was approved for clinical trials. As those skilled in the art will appreciate, the appropriate therapeutic dose level cannot be predicted a priori and depends on both the mechanism of action of the drug and the expression profile of the target antigen. As a result, while the on-target off-tumor toxicities of many gp100-specific vaccines and autologous T cell therapies that have been evaluated in the clinic are known, it remains impossible to predict a priori the appropriate therapeutic dose of T cell redirecting therapeutic agents. This is because vaccines and autologous T cell therapies generate only a small number of T cells in a patient that can recognize gp100-positive cells, whereas T cell redirecting therapeutic agents could theoretically give every CD3-positive T cell in a patient the ability to recognize and respond to gp100-positive cells. Summary of the Invention [Problem to be solved by the invention]

[0007] The safety and tolerability of IMCgp100 in patients with advanced melanoma was investigated in a phase I study (clinical trial identification number NCT01211262). Interim results of this study are presented (Middleton et al. (2015), Cancer Res 2015;75(15 Suppl):Abstract nr CT106). The maximum tolerated dose was determined to be 600 ng / kg administered once weekly, which resulted in a recommended phase II dose (RP2D) of 50 μg / dose, regardless of patient weight. During the dose-escalation part of the study, severe (grade 3 / 4) hypotension was observed as the main dose-limiting toxicity. Further episodes of severe hypotension were reported when a cohort of patients was subsequently dosed weekly at the RP2D. Other common adverse events included rash, pruritus, fever, and edema. Adverse events typically manifested shortly after the first or second dose. Further weekly dosing of IMCgp100 after two doses resulted in a reduction in adverse reactions and a general amelioration of the severity of these events. To increase tolerability during initial dosing, the first weekly dose of IMCgp100 was routinely reduced by 20% to 40 μg and dosed at RP2D for the following weeks. However, cases of severe hypotension continued to occur. Expression of gp100 in skin melanocytes provides a large reservoir of antigen-positive cells in the largest organ in the body. Potent targeting of gp100-positive cells by IMCgp100 leads to redirected T cell activity against gp100-positive skin melanocytes. This manifests as skin-related toxicity in patients. Analysis of patient serum and biopsy samples has demonstrated that local cytokine and chemokine release in both skin and tumors results in the very short-term movement of large numbers of T cells from the peripheral circulation into the tissues; the substantial fluid shift that accompanies this rapid T cell movement results in hypotension. [Means for solving the problem]

[0008] In a first aspect, the invention provides a bispecific therapeutic agent for redirecting T cells, comprising (i) a targeting moiety that binds to a YLEPGPVTA-HLA-A2 complex, and (ii) a CD3-binding T cell redirecting moiety fused to the targeting moiety, for use in a method of treating a gp100 positive cancer in a patient, the method comprising administering the bispecific therapeutic agent to a patient, wherein each dose is administered every 5 to 10 days, and wherein at least the first and second doses are 40 μg or less, and the second dose is higher than the first dose.

[0009] The inventors have surprisingly found an intrapatient dose escalation regimen that provides improved tolerability for the treatment of gp100-positive tumors with bispecific therapeutic agents that utilize a T cell redirection-mediated mechanism of action. The present invention provides an escalation dosing regimen that significantly reduces undesirable side effects (e.g., severe hypotension). This dosing regimen is useful for reducing the severity of on-target off-tumor toxicity in patients, thereby reducing the need for patients to remain hospitalized and / or in intensive care during the first few weeks of treatment. A further surprising benefit is that the use of this dosing regimen ameliorates the effects of on-target off-tumor on T cell activity, but allows patients to subsequently safely tolerate a higher overall therapeutic dose level of 50 μg than the existing R2P2, a level that is predicted to result in improved clinical efficacy. Without wishing to be bound by theory, the inventors believe that hypotension, a dose-limiting toxicity, is a potential risk factor for the C-terminal cytotoxicity of this bispecific therapeutic agent. max The hypothesis is that this is driven by the level of exposure, the presence of a large number of gp100-positive melanocytes in the skin compartment, and the extent of tumor gp100 expression in the patient, both in terms of the patient's disease burden and the expression level of gp100 in the patient's tumor. The inventors believe that this dosing regimen allows for sufficient destruction of normal skin melanocytes that express high levels of gp100, and subsequently allows for safer administration of higher therapeutic doses, resulting in improved targeting of the remaining gp100-positive tumor cells, thereby improving clinical efficacy.

[0010] The inventors found that patients with tumors of large diameter or high total disease burden experienced a desired tumor response at a higher total IMCgp100 exposure than patients with small tumors or disease burden. Based on this data, the inventors hypothesize that administering escalating drug doses for several weeks after reducing dose-limiting toxicity using the dosing regimen of the present invention will lead to enhanced tumor responses.

[0011] In the present invention, the bispecific therapeutic may be administered as follows: (a) at least one first dose in the range of 10 to 30 μg; (b) at least one second dose in the range of 20 to 40 μg, where each second dose is higher than the first dose; and then (c) at least one dose of at least 50 μg

[0012] In the present invention, each dose may be expressed as a specific weight of therapeutic agent, regardless of the patient's body weight or whether the same amount of therapeutic agent is administered when calculated by one of the other methods commonly used to calculate the appropriate dosage for a patient (e.g., weight of therapeutic agent per kg of body weight, body surface area, or lean muscle mass, etc.). The specific weight of therapeutic agent is preferably administered at weekly intervals, for example, on days 1, 8, 15, 22, etc. of the treatment regimen, although longer or shorter dosing intervals may be used. The dosing interval may depend on the antigen binding properties and clearance half-life of the bispecific therapeutic agent redirecting T cells. Thus, each dose may be administered every 6-9 days or every 6-8 days. Preferably, each dose is administered every 7 days. Each dose may be spaced apart at different intervals. Alternatively, each dose may be spaced apart at the same intervals.

[0013] The first dose may be in the range of 10-30 μg. The first dose may be in the range of 13-27 μg, 15-25 μg or 18-22 μg. The dose may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 μg. One suitable first dose is 20 μg, and this dose may be administered on a weekly basis. One first dose may be administered. Alternatively, two or more first doses may be administered, preferably two to five first doses, more preferably two first doses. When two or more first doses are administered, it is preferred that they are the same. However, they may be different.

[0014] The second dose may be in the range of 20-40 μg and may be higher than the first dose. The second dose may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μg higher than the first dose. The second dose may be in the range of 23-37 μg, 25-35 μg or 28-32 μg. Subsequent doses may be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 μg. One suitable second dose is 30 μg, and this dose may be administered on a weekly basis. One second dose may be administered. Alternatively, two or more second doses may be administered, preferably two to five second doses, more preferably two second doses. If two or more second doses are administered, it is preferred that they are the same. However, they may be different.

[0015] The dose after the second dose may be at least 50 μg. The dose may be in the range of 50-300 μg, 50-250 μg, 50-200 μg, 50-150 μg, 50-100 μg, 50-90 μg, 50-80 μg, 50-70 μg or 50-60 μg. The dose may be at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 μg. One preferred dose after the second dose is 50 μg. Another dose is 60 μg and yet another dose is 70 μg. The same dose may be used thereafter. Alternatively, the dose may be gradually increased. For example, the dose may be 5, 10, 15, 20, 30, 40 or 50 μg higher. The first dose may be 20 μg, the second dose may be 30 μg, and / or subsequent doses may be 50 μg or more.

[0016] A T cell redirecting bispecific therapeutic agent useful in the present invention comprises (i) a targeting moiety that binds to the YLEPGPVTA-HLA-A2 complex, and (ii) a CD3-binding T cell redirecting moiety fused to the targeting moiety. The targeting moiety may be a T cell receptor (TCR). The TCR is described using the International Immunogenetics (IMGT) TCR nomenclature and is linked to the IMGT public database of TCR sequences. The unique sequences defined by the IMGT nomenclature are widely known and available to those skilled in the art of TCRs. For example, they can be found in "T cell Receptor Factsbook" (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8; Lefranc (2011), Cold Spring Harb Protoc 2011(6):595-603; Lefranc (2001), Curr Protoc Immunol Appendix 1: Appendix 1O; Lefranc (2003), Leukemia 17(1):260-266 and the IMGT website (www.IMGT.org).

[0017] TCRs useful in the present invention may be in any form known to those skilled in the art. For example, the TCR may be an αβ heterodimer or in a single chain format (e.g. as described in WO9918129). Single chain TCRs include αβTCR polypeptides of Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ or Vα-Cα-L-Vβ-Cβ types, optionally inverted, where Vα and Vβ are TCR α and β variable regions, respectively, Cα and Cβ are TCR α and β constant regions, respectively, and L is a linker sequence. The TCR is preferably in soluble form (i.e. without transmembrane or cytoplasmic domains). For stability, such soluble TCRs preferably have disulfide bonds introduced between residues of the respective constant domains, for example as described in WO 03 / 020763. Suitable TCRs of this type include those which have a TRAC1 constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, except that Thr 48 of TRAC1 and Ser 57 of TRBC1 or TRBC2 are replaced with cysteine ​​residues which form a disulfide bond between the TRAC1 constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR. However, the TCR may also comprise full length α and β chains.

[0018] The α-chain and / or β-chain constant domains may be truncated compared to the native / naturally occurring TRAC / TRBC sequence. In addition, TRAC / TRBC may contain modifications. For example, the α-chain extracellular sequence may contain a modification relative to native / naturally occurring TRAC, in which amino acid T48 (according to IMGT numbering) of TRAC is replaced with C48. Similarly, the β-chain extracellular sequence may contain a modification relative to native / naturally occurring TRBC1 or TRBC2, in which S57 (according to IMGT numbering) of TRBC1 or TRBC2 is replaced with C57. These cysteine ​​substitutions in the native α-chain and β-chain extracellular sequences allow the formation of non-native interchain disulfide bonds that stabilize the refolded soluble TCR, i.e. the TCR formed by refolding the extracellular α-chain and β-chain (WO 03 / 020763). This non-native disulfide bond facilitates the display of correctly folded TCRs on phage (Li et al., Nat Biotechnol 2005 Mar;23(3):349-54). In addition, the use of stable disulfide-linked soluble TCRs allows for more convenient assessment of binding affinity and binding half-life. Alternative positions for forming non-native disulfide bonds are described in WO 03 / 020763.

[0019] TCRs useful in the present invention may be engineered to contain mutations. Methods for generating mutated high affinity TCR variants are known to those skilled in the art, for example phage display and site-directed mutagenesis (see, for example, WO 04 / 044004 and Li et al., Nat Biotechnol 2005 Mar;23(3):349-54). A TCR useful in the invention may have a binding affinity and / or binding half-life with respect to the YLEPGPVTA-HLA-A2 complex that is at least twice that of a TCR having an extracellular α chain sequence of SEQ ID NO: 2 and an extracellular β chain sequence of SEQ ID NO: 3. A TCR useful in the invention may have a K with respect to the complex of ≦8 μM, ≦5 μM, ≦1 μM, ≦0.1 μM, ≦0.01 μM, ≦0.001 μM or ≦0.0001 μM. Dand / or a binding half-life (T1 / 2) of ≥ 1.5s, ≥ 3s, ≥ 10s, ≥ 20s, ≥ 40s, ≥ 60s, ≥ 600s or ≥ 6000s. D and / or binding half-life (T1 / 2) of about 10-100 pM and about 6-48 hours, respectively. D and / or the binding half-life (T1 / 2) is about 24 pM and about 24 hours.

[0020] Binding affinity (equilibrium constant K D The binding half-life (denoted as T1 / 2) and binding half-life (denoted as T1 / 2) can be measured by any suitable method. D It is understood that T1 / 2 is ln2 / dissociation rate (k off ) Therefore, if T1 / 2 doubles, k off becomes 1 / 2. K of TCR D value and k off Values ​​are usually measured for soluble forms of the TCR, i.e. those truncated to remove hydrophobic transmembrane domain residues. A given TCR should therefore be understood to fulfil the requirements if its soluble form fulfils the requirements of having a binding affinity and / or binding half-life with respect to the YLEPGPVTA-HLA-A2 complex. Preferably, the binding affinity or binding half-life of a given TCR is measured several times (e.g. three or more times) using the same assay protocol and the results are averaged. In one preferred embodiment, these measurements are performed using the surface plasmon resonance (BIAcore) method of Example 3 of WO2011 / 001152. The reference gp100 TCR - a TCR with an extracellular α-chain sequence of SEQ ID NO: 2 and an extracellular β-chain sequence of SEQ ID NO: 3 - has a K of about 19 μM when measured by said method. D and about 1s -1 k off (i.e., T1 / 2 of about 0.7 s).

[0021] The TCR useful in the present invention may be linked to an anti-CD3 antibody, or a functional fragment or variant of said anti-CD3 antibody, as described in WO2011 / 001152. Antibody fragments and variants / analogues suitable for use in the compositions and methods described herein include minibodies, Fab fragments, F(ab')2 fragments, dsFv and scFv fragments, nanobodies, TM These include, but are not limited to, (commercially available from Ablynx, Belgium, constructs containing synthetic single immunoglobulin variable heavy chain domains derived from camelid (e.g. camel or llama) antibodies) and domain antibodies (Domantis, Belgium; which contain affinity matured single immunoglobulin variable heavy chain domains or immunoglobulin variable light chain domains).

[0022] TCR-anti-CD3 fusions useful in the present invention include the following: (i) the TCR α chain sequence of SEQ ID NO:2 in which amino acids 1 to 109 are replaced with the sequence of SEQ ID NO:4 and the amino acid at position 1 is S; (ii) the TCR α chain sequence of SEQ ID NO:2 in which amino acids 1 to 109 are replaced with the sequence of SEQ ID NO:4 and the amino acid at position 1 is A; (iii) the TCR α chain sequence of SEQ ID NO:2 in which amino acids 1 to 109 are replaced with the sequence of SEQ ID NO:4 and the amino acid at position 1 is G; (iv) the TCR α chain sequence of SEQ ID NO:2, in which amino acids 1 to 109 are replaced by the sequence of SEQ ID NO:4 and the amino acid at position 1 is S, and the C-terminus of the α chain is shortened by 8 amino acids from F196 to S203 based on the numbering of SEQ ID NO:2; (v) the TCR α chain sequence of SEQ ID NO:2, in which amino acids 1 to 109 are replaced with the sequence of SEQ ID NO:4 and the amino acid at position 1 is A, and the C-terminus of the α chain is truncated by 8 amino acids from F196 to S203 based on the numbering of SEQ ID NO:2; (vi) The TCR α chain sequence of SEQ ID NO: 2, in which amino acids 1 to 109 are replaced with the sequence of SEQ ID NO: 4 and the amino acid at position 1 is G, and the C-terminus of the α chain is shortened by 8 amino acids from F196 to S203 based on the numbering of SEQ ID NO: 2. and a TCR alpha chain amino acid sequence selected from the group consisting of: (vii) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, where the amino acids at positions 1 and 2 are D and I, respectively; (viii) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, where the amino acids at positions 1 and 2 are A and I, respectively; (ix) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, where the amino acids at positions 1 and 2 are A and Q, respectively; (x) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are D and I, respectively, the amino acids at positions 108 to 131 are substituted with RTSGPGDGGKGGPGKGPGGEGTKGTGPGG (SEQ ID NO:6), and the amino acids at positions 254 to 258 are substituted with GGEGGGSEGGGS (SEQ ID NO:7); (xi) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are D and I, respectively, the amino acid at position 257 is S, and the amino acid at position 258 is G; (xii) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are D and I, respectively, the amino acid at position 256 is S, and the amino acid at position 258 is G; (xiii) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are D and I, respectively, the amino acid at position 255 is S, and the amino acid at position 258 is G; (xiv) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, wherein the amino acids at positions 1 and 2 are A and Q, respectively, the amino acid at position 257 is S, and the amino acid at position 258 is G; (xv) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, wherein the amino acids at positions 1 and 2 are A and Q, respectively, the amino acid at position 256 is S, and the amino acid at position 258 is G; (xvi) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, wherein the amino acids at positions 1 and 2 are A and Q, respectively, the amino acid at position 255 is S, and the amino acid at position 258 is G; (xvii) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, wherein the amino acids at positions 1 and 2 are A and I, respectively, the amino acid at position 257 is S, and the amino acid at position 258 is G; (xviii) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, wherein the amino acids at positions 1 and 2 are A and I, respectively, the amino acid at position 256 is S, and the amino acid at position 258 is G; (xix) TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are A and I, respectively, the amino acid at position 255 is S, and the amino acid at position 258 is G. The TCR β chain-anti-CD3 amino acid sequence is selected from the group consisting of:

[0023] Examples of such TCR-anti-CD3 fusions are the α chain amino acid sequence is (i) and the β chain-anti-CD3 amino acid sequence is (vii); the α chain amino acid sequence is (i) and the β chain-anti-CD3 amino acid sequence is (x); the α chain amino acid sequence is (vi) and the β chain-anti-CD3 amino acid sequence is (ix); the α chain amino acid sequence is (v) and the β chain-anti-CD3 amino acid sequence is (viii); the α chain amino acid sequence is (vi) and the β chain-anti-CD3 amino acid sequence is (vii); the α chain amino acid sequence is (i) and the β chain-anti-CD3 amino acid sequence is (xi); the α chain amino acid sequence is (i) and the β chain-anti-CD3 amino acid sequence is (xii); the α chain amino acid sequence is (i) and the β chain-anti-CD3 amino acid sequence is (xiii); the α chain amino acid sequence is (vi) and the β chain-anti-CD3 amino acid sequence is (xiv); the α chain amino acid sequence is (vi) and the β chain-anti-CD3 amino acid sequence is (xv); the α chain amino acid sequence is (vi) and the β chain-anti-CD3 amino acid sequence is (xvi); the α chain amino acid sequence is (v) and the β chain-anti-CD3 amino acid sequence is (xvii); the α chain amino acid sequence is (v) and the β chain-anti-CD3 amino acid sequence is (xviii); the α chain amino acid sequence is (v) and the β chain-anti-CD3 amino acid sequence is (xix); the α chain amino acid sequence is (vi) and the β chain-anti-CD3 amino acid sequence is (xi); the α chain amino acid sequence is (vi) and the β chain-anti-CD3 amino acid sequence is (xii); and the α chain amino acid sequence is (vi) and the β chain anti-CD3 amino acid sequence is (xiii); It's TCR.

[0024] One suitable TCR-anti-CD3 fusion has the α-chain and β-chain of SEQ ID NOs: 4 and 5, respectively. One suitable TCR-anti-CD3 fusion has the α-chain amino acid sequence of (v) and the β-chain-anti-CD3 amino acid sequence of (viii). Alternatively, the targeting moiety may be an antibody. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules (i.e., molecules that contain an antigen-binding site that specifically binds an antigen, whether natural or partially or wholly synthetic). The term "antibody" includes antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanized antibodies (including any polypeptide comprising an immunoglobulin binding domain), any polypeptide or protein having a binding domain that is or is homologous to an antibody binding domain, whether natural or partially or wholly synthetic. Thus, chimeric molecules comprising an immunoglobulin binding domain or equivalent fused to another polypeptide are also included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023. Humanized antibodies may be modified antibodies having the variable regions of a non-human (e.g., murine) antibody and the constant regions of a human antibody. Methods for making humanized antibodies are described, for example, in U.S. Patent No. 5,225,539. Examples of antibodies include immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and subclasses of these isotypes; fragments comprising an antigen-binding domain, such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. Monoclonal antibodies may also be referred to herein as "mAbs."

[0025] It is possible to take an antibody, e.g. a monoclonal antibody, and use recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region or the complementarity determining regions (CDRs) of an antibody to the constant regions or constant regions and framework regions of a different immunoglobulin (see, e.g., EP-A-184187, GB 2188638A or EP-A-239400). Hybridomas (or other cells which produce antibodies) may be subjected to genetic mutations or other changes which may or may not alter the binding specificity of the antibody produced.

[0026] It has been shown that fragments of a whole antibody can function to bind antigens. Examples of binding fragments include (i) a Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of one VH domain (Ward, ES et al., Nature. 1989 Oct 12;341(6242):544-6); (v) an isolated CDR region; (vi) an F(ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single chain Fv molecule (scFv), in which the VH and VL domains are linked by a peptide linker which allows the two domains to associate to form an antigen-binding site (Bird et al., Science. 1988 Oct 21;242(4877):423-6; Huston et al., Proc Natl Acad Sci US A. 1988 Aug;85(16):5879-83;(viii) bispecific single chain Fv dimers (PCT / US92 / 09965); and (ix) "diabodies", multivalent or multispecific fragments constructed by genetic fusion (WO94 / 13804; P. Hollinger et al., Proc Natl Acad Sci US A. 1993 Jul 15;90(14):6444-8). Diabodies are multimers of polypeptides in which each polypeptide comprises a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen-binding site, where the antigen-binding site is formed by association of a first domain of one polypeptide in the multimer with a second domain of another polypeptide in the multimer (WO94 / 13804).When bispecific antibodies are used, they may be conventional bispecific antibodies, which can be produced in a variety of ways (Hollinger & Winter, Curr Opin Biotechnol. 1993 Aug;4(4):446-9) (e.g., produced chemically or from hybrid hybridomas), or may be any of the bispecific antibody fragments described above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFvs can be constructed without the Fc region, using only the variable domains, presumably reducing the effects of anti-idiotypic reaction. Other forms of bispecific antibodies include the single chain "Janusins" described by Traunecker et al., EMBO J. 1991 Dec;10(12):3655-9. Bispecific diabodies, which are not bispecific whole antibodies, may also be useful because they can be easily constructed and expressed in E. coli. Diabodies (and many other polypeptides, e.g., antibody fragments) of appropriate binding specificity can be readily selected from libraries using phage display (WO94 / 13804). If one arm of the diabody is to be kept constant, e.g., specific for antigen X, a library can be made in which the other arm is varied to select antibodies of appropriate specificity. An "antigen-binding domain" is a portion of an antibody that contains an area that specifically binds to and is complementary to part or all of an antigen. In cases where the antigen is large, an antibody may only bind to a particular part of the antigen (called an epitope). An antigen-binding domain may be provided by one or more antibody variable domains. The antigen-binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0027] The binding moiety may be a TCR-like molecule designed to specifically bind to a peptide-MHC complex. Particularly preferred are TCR mimetic antibodies (e.g., those described in WO2007143104 and Sergeeva et al., Blood. 2011 Apr 21;117(16):4262-72 and / or Dahan and Reiter, Expert Rev Mol Med. 2012 Feb 24;14:e6). Binding moieties based on engineered protein scaffolds are also included in the present invention. Protein scaffolds are derived from stable, soluble, naturally occurring protein structures that have been modified to provide binding sites for target molecules of interest. Examples of engineered protein scaffolds include, but are not limited to, affibodies, which are based on the Z domain of Staphylococcus protein A and provide a binding interface on two alpha helices (Nygren, FEBS J. 2008 Jun;275(11):2668-76); anticalins, which are derived from lipocalins and incorporate a binding site for small ligands at the open end of a beta barrel fold (Skerra, FEBS J. 2008 Jun;275(11):2677-83), nanobodies and DARPins. Engineered protein scaffolds are typically targeted to bind the same antigenic proteins as antibodies and are potential therapeutics. They may act as inhibitors or antagonists, or may act in vivo as delivery vehicles for target molecules, e.g. toxins, to specific tissues (Gebauer and Skerra, Curr Opin Chem Biol. 2009 Jun;13(3):245-55). Short peptides may also be used to bind target proteins. Phylomers are natural structured peptides derived from bacterial genomes. The peptides represent diverse protein structural folds and can be used to inhibit / disrupt protein-protein interactions in vivo (Watt, Nat Biotechnol. 2006 Feb;24(2):177-83). As described above for the TCR, the CD3-binding T cell redirecting moiety may be an anti-CD3 antibody, or a functional fragment or variant of said anti-CD3 antibody, and may be as described in WO2011 / 001152.

[0028] In one embodiment of the invention, the method comprises administering to a patient a dose of 20 μg or less of a T cell redirecting bispecific therapy in week 1, a dose of 30 μg or less in week 2, and a dose of at least 50 μg in weeks 3 and thereafter, wherein the TCR portion of the bispecific therapy is linked to a T cell redirecting anti-CD3 antibody or fragment thereof and has a binding affinity and / or binding half-life for the YLEPGPVTA-HLA-A2 complex that is at least twice that of a TCR having an extracellular α chain sequence of SEQ ID NO:2 and an extracellular β chain sequence of SEQ ID NO:3. The T cell redirecting bispecific therapeutic agent is preferably administered by intravenous infusion. Alternative routes of administration include other parenteral routes, such as subcutaneous or intramuscular injection, enteral routes (including oral or rectal routes), inhalation or intranasal routes.

[0029] The bispecific therapeutic agents used in the present invention may be administered as monotherapy. Alternatively, the bispecific therapeutic agents may be administered in combination with one or more anti-cancer therapeutic agents, preferably immunomodulatory therapeutic agents. The possibility of further immune activation with combination therapy increases the risk of on-target but off-tumor toxicity. Such therapeutic agents include: Chemotherapeutic agents, such as dacarbazine and temozolomide, Immunotherapeutic agents, such as interleukin-2 (IL-2) and interferon (IFN) Checkpoint inhibitors, such as agents that target PD-1 or PD-L1, such as pembrolizumab, nivolumab, atezolizumab, avelumab, and durvalumab, and agents that target CTLA-4, such as ipilimumab and tremelimumab; BRAF inhibitors, such as vemurafenib and dabrafenib, * MEK inhibitors, e.g., trametinib, TGF-β inhibitors, e.g. galunisertib, MET kinase inhibitors, for example, merestinib.

[0030] Suitable combination therapies use durvalumab, tremelimumab, galunisertib and merestinib in combination with the T cell redirecting bispecific therapeutics listed above. When merestinib (see WO2010011538; or similar amidophenoxyindazoles described therein) is used in combination with the bispecific therapeutic, the dose of merestinib can be in the range of 40-120 mg once daily, preferably in the range of 80-120 mg once daily. The bispecific therapy may be administered alone for the first and subsequent doses and then combined with an additional therapy, or vice versa. In embodiments of the invention in which a T cell redirecting bispecific therapy and another cancer therapy are administered in combination, the T cell redirecting bispecific therapy may be administered alone in weeks 1 and 2, with the other therapy added in weeks 3 and beyond. Combination therapy may lead to increased immune activation, including cytokine and chemokine secretion and lymphocyte migration, and thus increased risk of side effects (e.g., severe hypotension). Thus, the T cell redirecting bispecific therapeutic agent may be initially administered as a single agent prior to combination administration. In one preferred embodiment, a dose of 20 μg or less is administered in the first week, a dose of 30 μg or less is administered in the second week, and a dose of at least 50 μg is administered in the third week and thereafter. One or more additional immunomodulatory therapeutic agents may be administered starting from the third week.

[0031] The present invention relates to the treatment of gp100 positive cancers. One such cancer is melanoma. Malignant melanoma continues to be a major public health challenge worldwide. In the United States, it is estimated that nearly 76,000 new cases of melanoma were diagnosed in 2016, and nearly 10,000 deaths from melanoma occurred (American Cancer Society Statistics, 2016). In addition, the diagnosis rate of melanoma continues to rise at a substantial rate; according to 2015 Surveillance, Epidemiology, and End Results data, the average incidence has increased 1.4% annually in the last decade, and melanoma is now the fifth most commonly diagnosed cancer in the United States. While the prognosis for patients with early stage disease is good after surgical resection, the median survival rate declines rapidly with disease progression, dropping to less than one year for patients with distant metastatic disease (Garbe et al., Oncologist 2011;16:5-24). The rising incidence of melanoma, coupled with poor outcomes with standard chemotherapy and early signs of immune-based therapy, has led to intensive research into novel approaches and combinations to enhance anti-tumor immune responses.

[0032] The melanoma to be treated according to the present invention may be cutaneous or uveal melanoma (also known as intraocular melanoma), or may be any one of lentigo maligna, superficial spreading melanoma, acral lentigo, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft tissue melanoma, small cell melanoma with small nevus-like cells, and Spitz nevus-like melanoma. The melanoma to be treated is generally late stage disease (advanced disease), typically characterized by metastatic disease, e.g., stage III and / or stage IV disease (Balch et al. J Clin Oncol 2009;27:6199-206).

[0033] Uveal melanoma (UM) is a rare type of melanoma that is biologically distinct from cutaneous melanoma. UM is a highly malignant neoplasm that affects the vascular layers of the eye (iris, ciliary body, and choroid). Its incidence is rare (approximately 3% of melanoma cases, corresponding to approximately 4,000 cases / year worldwide) (Papastefanou et al. (2011), J Skin Cancer;2011:573974). The suppressive environment in UM and the lack of activity of checkpoint inhibition suggest that recruitment of tumor-specific activated T cells may have antitumor activity in this disease setting. As mentioned above, other non-melanoma cancers have also been reported in the literature to be gp100 positive, examples of which include clear cell sarcoma and neurological cancers (e.g., some gliomas). The present invention can also be used to treat such cancers.

[0034] In a further aspect, the invention provides a method of treating gp100 positive cancer in a patient comprising administering to the patient a bispecific therapy that redirects T cells, each dose being administered every 5 to 10 days, wherein at least a first and second dose is 40 μg or less, and wherein the second dose is higher than the first dose.

[0035] In a further aspect, the present invention provides a T cell redirecting bispecific therapeutic comprising (i) a targeting moiety that binds to the YLEPGPVTA-HLA-A2 complex, and (ii) a CD3-binding T cell redirecting moiety to which said targeting moiety is fused, comprising the sequential steps of: (a) administering to a patient a weekly dose of a bispecific therapeutic agent in the range of 10 to 30 μg; (b)(i) if the patient experiences treatment-related hypotension of Grade 1 or less, increasing the dose of the bispecific therapy administered to the patient to a weekly dose of between 20 and 40 μg; (b)(ii) if the patient experiences treatment-related hypotension of Grade 2 or greater, continuing to administer to the patient a weekly dose of the bispecific therapeutic in the range of 10-30 μg until the patient experiences treatment-related hypotension of Grade 1 or less, thereafter increasing the dose administered to the patient to a weekly dose of the bispecific therapeutic in the range of 20-40 μg; and (c)(i) if the patient experiences Grade 1 or less treatment-related hypotension, increasing the dose administered to the patient to a weekly dose of at least 50 μg of the bispecific therapy; (c)(ii) if the patient experiences treatment-related hypotension of Grade 2 or greater, continuing to administer to the patient a weekly dose of the bispecific therapeutic agent in the range of 20-40 μg until the patient experiences treatment-related hypotension of Grade 1 or less, and then increasing the dose administered to the patient to a weekly dose of at least 50 μg of the bispecific therapeutic agent. The present invention provides a T cell retargeting bispecific therapeutic agent for use in a method for treating gp100 positive cancer in a patient, including those

[0036] In this respect, the method further comprises, between step (b) and step (c), the step of: (d)(i) if the patient experiences Grade 1 or less treatment-related hypotension, increasing the patient's dose to a weekly dose of the bispecific therapy in the range of 30 to 45 μg; (d)(ii) if the patient experiences treatment-related hypotension of Grade 2 or greater, continuing to administer to the patient a weekly dose of the bispecific therapeutic agent in the range of 20-40 μg until the patient experiences treatment-related hypotension of Grade 1 or less, and then increasing the dose administered to the patient to a weekly dose of the bispecific therapeutic agent in the range of 30-45 μg. It may further include. The grade of hypotension can be assessed by a physician.

[0037] Preferred features of each aspect of the invention are as for each of the other aspects, mutatis mutandis. It is understood that certain features of the invention (which for clarity are described in the context of separate embodiments) may be provided in combination in a single embodiment. Conversely, various features of the invention (which for brevity are described in the context of a single embodiment) may be provided separately or in any suitable subcombination. All combinations of the embodiments relating to the T cell redirecting bispecific therapeutic agent for use in and methods of treating gp100 positive cancer are specifically embraced by the present invention and are described herein as if each and every combination was individually and explicitly disclosed. Publications referred to herein are incorporated herein to the fullest extent permitted by law. Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. In the following, reference is made to the accompanying drawings. [Brief description of the drawings]

[0038] [Figure 1] FIG. 1 shows the amino acid sequence of the α chain extracellular domain of the reference gp100 TCR (SEQ ID NO:2); [Diagram 2] FIG. 2 shows the amino acid sequence of the β chain extracellular domain of the reference gp100 TCR (SEQ ID NO:3); [Diagram 3] FIG. 3 shows the amino acid sequence of the gp100-specific TCR α chain (SEQ ID NO:4); [Figure 4] FIG. 4 shows the amino acid sequence (SEQ ID NO:5) of an anti-CD3 scFv antibody fragment (bold) fused to the N-terminus of a gp100-specific TCR β chain via a linker, i.e., GGGGS (underlined); [Diagram 5] Figure 5 shows the occurrence of toxicity (severe and / or profound hypotension) versus number of doses of IMCgp100; [Figure 6] FIG. 6 shows lymphocyte migration from the periphery after administration of the first dose of IMCgp100; [Figure 7]Figure 7 shows T cell proliferation by IMCgp100 in the presence or absence of anti-CTLA-4; [Figure 8] Figure 8 shows T cell proliferation by IMCgp100 in the presence or absence of anti-PD-L1; [Figure 9] FIG. 9 shows the secretion of cytokines IFNγ, TNFα and IL-2 by IMCgp100 in the presence or absence of anti-CTLA-4; and [Figure 10] FIG. 10 shows secretion of the cytokine IFNγ by IMCgp100 in the presence or absence of anti-CTLA-4. EXAMPLES

[0039] Working Example Example 1 - Identification of a dosing regimen of IMCgplOO that ameliorates drug-associated severe hypotension and allows for an increase in the upper dose limit IMCgp100 is the YLEPGPVTA peptide-HLA-A * IMCgp100 is a T cell redirecting bispecific agent comprising a soluble affinity-enhanced TCR that binds to the 02 complex and fused to an anti-CD3 scFv [the α and β chains of IMCgp100 are SEQ ID NOs: 4 and 5, respectively]. IMCgp100 was investigated in a first-in-human (FIH) open-label dose-ranging study to evaluate the safety and tolerability of IMCgp100 in patients with advanced malignant melanoma (Clinical Trial Identification Number: NCT01211262). This study was designed to identify the maximum tolerated dose (MTD) or recommended phase II dose (RP2D) of IMCgp100 in two repeated dosing regimens: (arm 1) weekly dosing (RP2D-QW) and (arm 2) daily dosing x 4 days (RP2D-QD).

[0040] Patients with stage IV or unresectable stage III malignant melanoma were enrolled in this study. All patients were HLA-A *Patients were required to have BRCA-positive BRCA2, be aged 18 years or older, be classified as having measurable disease according to RECIST 1.1 criteria, have an estimated life expectancy of more than 3 months, and have an Eastern Cooperative Oncology Group (ECOG) performance status of ≤1. There was no limit to the number of previous therapies. Patients were evaluated for adequate hematologic, renal, hepatic, and cardiac function. Patients with symptomatic brain metastases were excluded from the study. A total of 84 patients were treated. IMCgp100 was administered by intravenous infusion using a controlled infusion pump. Screening procedures and tests to establish eligibility were performed within 14 days prior to initiation of IMCgp100 dosing, with the exception of HLA typing, MRI scan, ophthalmological, audiological and echocardiographic examinations, which were performed within 28 days prior to dosing initiation. Informed consent was obtained from all patients. Blood samples for hematological, biochemical and pharmacokinetic (PK) analyses were obtained at screening, on day 1 prior to infusion, 4, 10 and 24 hours, and on days 2, 8 and 30 after initiation of infusion.

[0041] Once-weekly dosing: consequences of dose escalation The maximum tolerated dose (MTD) was defined according to a standard 3+3 Phase I dose escalation protocol. Briefly, the dose of IMCgp100 was escalated in cohorts of 3 (+3) patients until the MTD criteria were met or the target limiting dose was reached. Dose escalation was in 3-fold increments and transitioned to a modified Fibonacci protocol according to the safety and PK profile or if dose-limiting toxicity (DLT) was reported. The MTD was defined as the highest dose level at which DLT occurred in ≥33% of enrolled patients. DLTs were observed within an 8-day post-treatment window and were assessed according to the NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Transient grade 3 or grade 4 lymphopenia and non-fatal skin rash were excluded as dose-limiting criteria due to the expected pharmacological effects of the drug.

[0042] During dose escalation, 31 patients were treated in 8 dose cohorts, receiving 5 ng / kg to 900 ng / kg of drug. Grade 3 or 4 dose-limiting toxicity (DLT) was observed in 4 patients at 405 ng / kg (n=1 of 6), 600 ng / kg (n=1 of 6), and 900 ng / kg (n=2 of 6) during dose escalation. In 3 of the 4 DLT cases of grade 3 or 4 hypotension, the events occurred approximately 12-18 hours after dosing with the first dose of IMCgp100; one DLT hypotensive event occurred with the second dose at 405 ng / kg in a patient receiving concurrent antihypertensive therapy. These four cases of hypotension were managed with IV fluids (normal saline and colloid infusion); none of the patients required pharmacologic (inotropic) support for blood pressure, and all were treated with IV corticosteroid therapy. All cases of grade 3 or 4 hypotension resolved within 2 days with fluid therapy and IV corticosteroid therapy.

[0043] [Table 1] Based on this data, the MTD for once weekly dosing was determined to be 600 ng / kg.

[0044] Once-weekly dosing: Results from the RP2D-QW cohort Review of PK and safety data from the weekly dose-escalation cohorts suggested that more severe toxicity and higher drug exposure of IMCgp100 were associated with higher absolute doses. Based on these data and the range of absolute doses (n=5 patients, range 34-66 μg QW, median dose 54 μg) with an MTD of 600 ng / kg, the recommended phase 2 dose for the weekly dosing regimen (referred to as RP2D-QW) was initially determined to be a flat dose of 50 μg administered once weekly.

[0045] During this period, three patients experienced adverse events (including hypotension grade 2 or higher). These events were noted to be limited to the first two doses (e.g., day 1 of cycle 1 (C1D1) and day 8 of cycle 1 (C1D8)) (Figure 5). In an attempt to avoid severe hypotensive events, the first dose of IMC gp100 was subsequently reduced to 40 μg; however, hypotension (grade 3 / 4) was observed after dosing with C1D1 and / or C1D8 in two patients; one patient also experienced grade 2 hypotension after dosing with the third dose. No cases of severe hypotension were reported after subsequent doses. It should be noted that the inventors found a link between the frequency and severity of hypotension and gp100 expression levels in patient tumors, with patients with uveal melanoma having particularly high gp100 expression levels and therefore a higher risk of toxicity.

[0046] Seven patients (six with uveal melanoma and one with cutaneous melanoma) at high risk of toxicity due to high gp100 expression levels in their tumors were enrolled in a weekly dose expansion phase in which they received a first dose of 20 μg (C1D1), followed by a second dose of 30 μg (C1D8), and finally a flat dose of 50 μg from the third dose onwards. Surprisingly, no severe (grade 3 / 4) hypotension was reported in these seven patients, despite their high risk of developing gp100 expression-mediated toxicity. Table 2 below summarizes the three dosing regimens used in this study, the number of patients treated with each regimen, and the number of hypotensive adverse events reported.

[0047] [Table 2] These data demonstrate the surprising benefit of a two-stage intrapatient dose-escalation regimen for remission of drug-associated severe hypotension when treating patients with gp100-positive cancer.

[0048] Hypotension is due to lymphocyte migration and cytokine release Blood analysis of a subset of patients dosed at weekly RP2D showed significant lymphocyte migration following IMCgp100 administration. Lymphocyte levels declined substantially approximately 12-24 hours after infusion, returning to baseline levels by day 8. This effect was more pronounced in cases with severe and / or profound hypotension (Figure 6). Cytokine analysis revealed modest levels of one or more inflammatory cytokines. The largest increase observed in the periphery was in the levels of tissue chemoattractant chemokines. This paralleled a transient decline in peripheral circulating lymphocytes, which were nearly gone by 48 hours after dosing and reached pre-dosing levels by day 8.

[0049] Further validation of intra-patient dosing regimens Retrospective review of tumor response data from patients treated in the above Phase 1 trials noted that, in general, the desired response was obtained in patients receiving higher absolute doses (approximately 65-85 μg). Therefore, we hypothesized that using 20 μg in C1D1 and 30 μg in C1D8 would allow the use of higher absolute doses, i.e., 50 μg or more, from cycle 1 day 15 (C1D15) onwards, possibly improving efficacy. To validate this, IMCgp100 is being further investigated in patients with advanced uveal melanoma in an ongoing Phase I open-label multicenter study (clinical trial identification number: NCT02570308). The first part of the study follows a standard 3+3 dose escalation design. The first patient cohort is receiving IMCgp100 by intravenous infusion at a fixed dose of 20 μg on C1D1, 30 μg on C1D8, followed by 60 μg from C1D15. Initial data indicate that no severe hypotension-related events were reported for patients in this cohort (n=3). Further patient cohorts are planned to receive higher doses (e.g., 70 μg, 80 μg or more) on C1D15.

[0050] Example 2 - Evidence of increased risk of severe hypotension for IMCgp100 administered in combination with other immunomodulatory agents IMCgp100-mediated immune activation was examined in vitro in the presence or absence of checkpoint inhibitor antibodies against PD-L1 / PD-1 and CTLA-4. Combining IMCgp100 with anti-CTLA-4 enhances the potency of T cell responses In this experiment, T cell proliferation was used as an indicator of efficacy. IMCgp100 was used at a concentration of 80 pM in the presence or absence of 40 μg / ml of anti-CTLA-4 (clone L3D10; Biolegend). HLA-A was pulsed with 10 nM or 100 nM of gp100 peptide. * 02 positive monocytes were used as target antigen presenting cells and plated at 10,000 cells / well. Effector CD3+ T cells were labeled with Cell Tracker Violet. T cell proliferation was measured after 5 days using a FACS-based Intellicyt assay. The results shown in Figure 7 indicate that the combination of IMCgp100 and anti-CTLA-4 results in improved T cell responses (at lower peptide concentrations) compared to IMCgp100 alone, suggesting that patients treated with this combination may have higher levels of both efficacy and gp100-specific on-target and off-tumor toxicity than IMCgp100 monotherapy.

[0051] Combining IMCgp100 with anti-PD-L1 increases T cell killing efficacy In this study, T cell killing was used as an indicator of efficacy. IMCgp100 was used at concentrations of 80 pM and 100 pM in the presence or absence of 10 μg / ml anti-PD-L1 (clone 29E.2A3; BioLegend). * 02 positive Mel624 cells were used as target antigen presenting cells. CD8+ T cells were used as effector cells at an effector target ratio of 5:1. T cell killing was measured over 3 days using the Incucyte ZOOM assay. Caspase-3 / 7 activation in target cells was monitored as a measure of apoptosis (imaged every 2 hours). The results shown in Figure 8 indicate that the combination of IMCgp100 and anti-PD-L1 results in enhanced T cell killing compared to IMCgp100 alone, suggesting that in patients treated with this combination, both efficacy and gp100-specific on-target and off-tumor toxicity may be at a higher level than with IMCgp100 monotherapy.

[0052] Combining IMCgp100 with anti-CTLA-4 or anti-PD-L1 enhances pro-inflammatory cytokine production In this experiment, cytokine secretion was assessed using Meso Scale Discovery's V-PLEX Human Pro-Inflammatory Panel 1 Assay Kit. For combinations of IMCgp100 with anti-CTLA-4 or anti-PD-L1, supernatant samples were taken at 24 hours from the proliferation and killing assays, respectively. The data shown in FIG. 9 demonstrate that IFNγ, TNFα and IL-2 secretion (at low peptide concentrations) is enhanced in the presence of IMCgp100 and anti-CTLA-4 compared to IMCgp100 alone. The data shown in Figure 10 demonstrate that IFNγ secretion is enhanced in the presence of IMCgp100 and anti-PD-L1 compared to IMCgp100 alone. These data suggest that immune activation with IMCgp100 combination therapy is increased compared to IMCgp100 monotherapy, and therefore that patients receiving combination therapy may be at increased risk of severe hypotension due to on- and off-tumor targeting of gp100-positive melanocytes.

Claims

1. (i) a targeting moiety that binds to the YLEPGPVTA-HLA-A2 complex; and (ii) a CD3-binding T cell redirecting moiety fused to the targeting moiety, wherein each dose is administered every 5-10 days to deliver a T cell redirecting bispecific therapeutic to a patient. (a) a first dose in the range of 10 to 30 μg, followed by (b) a second dose in the range of 20 to 40 μg, where each second dose is higher than the first dose. and then (c) at least one dose of at least 60 μg 23. A T cell retargeting bispecific therapy for use in a method of treating a gp100 positive cancer in a patient comprising administering to said patient a T cell retargeting bispecific therapy.

2. 2. The bispecific therapeutic for use according to claim 1, wherein each dose is administered every 7 days.

3. 3. The bispecific therapeutic for use according to claim 1 or 2, wherein the first dose is 20 μg, the second dose is 30 μg, and / or the dose after the second dose is at least 60 μg.

4. The bispecific therapeutic for use according to any one of claims 1 to 3, wherein the second dose is administered twice.

5. The bispecific therapeutic for use according to any one of claims 1 to 4, wherein the targeting moiety is a T cell receptor (TCR).

6. 6. A bispecific therapeutic agent for use according to claim 5, wherein the TCR has a binding affinity and / or binding half-life for the YLEPGPVTA-HLA-A2 complex that is at least twice that of a TCR having an extracellular alpha chain sequence of SEQ ID NO:2 and an extracellular beta chain sequence of SEQ ID NO:

3.

7. The bispecific therapeutic for use according to any one of claims 1 to 6, wherein the CD3-binding T cell redirecting moiety is an anti-CD3 antibody.

8. the below described: (i) the TCR α chain sequence of SEQ ID NO:2 in which amino acids 1 to 109 are replaced by the sequence of SEQ ID NO:4 and the amino acid at position 1 is S; (ii) the TCR α chain sequence of SEQ ID NO:2 in which amino acids 1 to 109 are replaced with the sequence of SEQ ID NO:4 and the amino acid at position 1 is A; (iii) the TCR α chain sequence of SEQ ID NO:2 in which amino acids 1 to 109 are replaced with the sequence of SEQ ID NO:4 and the amino acid at position 1 is a G; (iv) the TCR α chain sequence of SEQ ID NO:2 in which amino acids 1 to 109 are replaced by the sequence of SEQ ID NO:4 and the amino acid at position 1 is S, and the C-terminus of the α chain is truncated by 8 amino acids from F196 to S203 based on the numbering of SEQ ID NO:2; (v) the TCR α chain sequence of SEQ ID NO:2 in which amino acids 1 to 109 are replaced by the sequence of SEQ ID NO:4 with the amino acid at position 1 being A, and the C-terminus of the α chain is truncated by 8 amino acids from F196 to S203 based on the numbering of SEQ ID NO:2; (vi) a TCR α chain sequence of SEQ ID NO:2, in which amino acids 1 to 109 are replaced with the sequence of SEQ ID NO:4 and the amino acid at position 1 is G, and the C-terminus of the α chain is truncated by 8 amino acids from F196 to S203 based on the numbering of SEQ ID NO:

2. and a TCR alpha chain amino acid sequence selected from the group consisting of: (vii) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are D and I, respectively; (viii) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are A and I, respectively; (ix) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are A and Q, respectively; (x) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are D and I, respectively, and amino acids at positions 108 to 131 are substituted with RTSGPGDGGKGGPGKGPGGEGTKGTGPGG (SEQ ID NO:6), and amino acids at positions 254 to 258 are substituted with GGEGGGSEGGGS (SEQ ID NO:7); (xi) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are D and I, respectively, the amino acid at position 257 is S, and the amino acid at position 258 is G; (xii) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are D and I, respectively, the amino acid at position 256 is S, and the amino acid at position 258 is G; (xiii) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are D and I, respectively, the amino acid at position 255 is S, and the amino acid at position 258 is G; (xiv) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are A and Q, respectively, the amino acid at position 257 is S, and the amino acid at position 258 is G; (xv) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are A and Q, respectively, the amino acid at position 256 is S, and the amino acid at position 258 is G; (xvi) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are A and Q, respectively, the amino acid at position 255 is S, and the amino acid at position 258 is G; (xvii) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are A and I, respectively, the amino acid at position 257 is S, and the amino acid at position 258 is G; (xviii) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, wherein the amino acids at positions 1 and 2 are A and I, respectively, the amino acid at position 256 is S, and the amino acid at position 258 is G; (xix) the TCR β chain-anti-CD3 sequence of SEQ ID NO:5, in which the amino acids at positions 1 and 2 are A and I, respectively, the amino acid at position 255 is S, and the amino acid at position 258 is G; 8. The bispecific therapeutic agent for use according to claim 6 or 7, comprising a TCR β chain-anti-CD3 amino acid sequence selected from the group consisting of:

9. the α chain amino acid sequence is (i) and the β chain-anti-CD3 amino acid sequence is (vii); the α chain amino acid sequence is (i) and the β chain-anti-CD3 amino acid sequence is (x); the α chain amino acid sequence is (vi) and the β chain-anti-CD3 amino acid sequence is (ix); the α chain amino acid sequence is (v) and the β chain anti-CD3 amino acid sequence is (viii); the α chain amino acid sequence is (vi) and the β chain-anti-CD3 amino acid sequence is (vii); the α chain amino acid sequence is (i) and the β chain-anti-CD3 amino acid sequence is (xi); the α chain amino acid sequence is (i) and the β chain-anti-CD3 amino acid sequence is (xii); the α chain amino acid sequence is (i) and the β chain-anti-CD3 amino acid sequence is (xiii); the α chain amino acid sequence is (vi) and the β chain-anti-CD3 amino acid sequence is (xiv); the α chain amino acid sequence is (vi) and the β chain-anti-CD3 amino acid sequence is (xv); the α chain amino acid sequence is (vi) and the β chain-anti-CD3 amino acid sequence is (xvi); the α chain amino acid sequence is (v) and the β chain-anti-CD3 amino acid sequence is (xvii); the α chain amino acid sequence is (v) and the β chain-anti-CD3 amino acid sequence is (xviii); the α chain amino acid sequence is (v) and the β chain anti-CD3 amino acid sequence is (xix); the α chain amino acid sequence is (vi) and the β chain-anti-CD3 amino acid sequence is (xi); the α chain amino acid sequence is (vi) and the β chain-anti-CD3 amino acid sequence is (xii); and the α chain amino acid sequence is (vi) and the β chain anti-CD3 amino acid sequence is (xiii); A bispecific therapeutic agent for use according to claim 8.

10. 10. The bispecific therapeutic agent for use according to claim 9, wherein the α chain amino acid sequence is (v) and the β chain-anti-CD3 amino acid sequence is (viii).

11. The bispecific therapeutic agent for use according to any one of claims 1 to 10, administered in combination with one or more anti-cancer therapeutic agents.

12. 12. The bispecific therapeutic for use according to claim 11, wherein the anti-cancer therapeutic is durvalumab, tremelimumab, galunisertib and merestinib.

13. 13. The bispecific therapeutic for use according to claim 12, wherein the anti-cancer therapeutic is merestinib and the dosage of merestinib is in the range of 40-120 mg once per day, preferably in the range of 80-120 mg once per day.

14. The bispecific therapeutic for use according to any one of claims 1 to 13, wherein the gp100 positive cancer is melanoma.

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