CN118715240A - HIV-specific binding molecules and TCR - Google Patents

Abstract

Specific binding molecules are provided, including T cell receptors (TCRs) that bind to the HLA-A*02 restricted peptide SLYNTVATL (SEQ ID NO: 1) derived from HIV Gag gene product p17. Relative to natural TCRs, the TCRs of the present invention comprise non-natural mutations within the α and/or β variable domains. The specific binding molecules of the present invention have improved stability and/or yield, but unexpectedly retain the favorable properties of the specific binding molecules from which they are derived. Such specific binding molecules are particularly useful in developing soluble immunotherapeutic agents for treating HIV-infected individuals.

Description

HIV specific binding molecules and TCRs

Disclosure of Invention

The present invention relates to specific binding molecules, such as T Cell Receptors (TCRs), which bind HLA-A x 02 restriction peptide SLYNTVATL (SEQ ID NO: 1) derived from the HIV Gag gene product p 17. The specific binding molecule may comprise a CDR sequence embedded within a framework sequence. The CDR and framework sequences may correspond to T Cell Receptor (TCR) variable domains, and may further comprise non-natural mutations relative to the native TCR variable domains. The specific binding molecules of the present invention have improved stability and/or yield and unexpectedly retain the advantageous properties of the specific binding molecules from which they are derived (high affinity, specificity and sensitivity for the complex of SEQ ID NO:1 and HLA-A-02 and drive particularly efficient T cell responses). Such specific binding molecules are particularly useful in the development of soluble immunotherapeutic agents for the treatment of HIV-infected individuals.

Disclosed herein are specific binding molecules having the property of binding SLYNTVATL (SEQ ID NO: 1) complexed with HLA-A-02 and comprising a TCR a chain variable domain and a TCR β chain variable domain, wherein the a chain variable domain comprises an amino acid sequence selected from the group consisting of:

(a)

(b)

Or (b)

(c)

Optionally with an N-terminal methionine (SEQ ID NOS: 41-43), and

The β chain variable domain comprises the following amino acid sequence:

optionally with an N-terminal methionine (SEQ ID NO: 44).

In some embodiments, the specific binding molecule has the property of binding SLYNTVATL (SEQ ID NO: 1) complexed with HLA-A x 02, and comprises a TCR a chain variable domain and a TCR β chain variable domain, wherein the a chain variable domain has an amino acid sequence selected from the group consisting of:

(a)

(b)

Or (b)

(c)

Optionally with an N-terminal methionine (SEQ ID NOS: 41-43), and/or

The β chain variable domain has the following amino acid sequence:

Optionally having an N-terminal methionine:

In another aspect, disclosed herein are nucleic acid molecules encoding a TCR a chain and/or a TCR β chain, wherein the TCR a chain comprises a variable domain amino acid sequence selected from the group consisting of seq id nos:

(a)

(b)

Or (b)

(c)

Optionally with an N-terminal methionine (SEQ ID NOS: 41-43), and/or

The TCR β chain comprises a variable domain amino acid sequence:

optionally with an N-terminal methionine (SEQ ID NO: 44).

In some embodiments, the nucleic acid molecule encodes a TCR a chain and/or a TCR β chain, wherein the TCR a chain has a variable domain amino acid sequence selected from the group consisting of:

(a)

(b)

Or (b)

(c)

Optionally with an N-terminal methionine (SEQ ID NOS: 41-43), and/or

The TCR β chain has a variable domain amino acid sequence:

optionally with an N-terminal methionine (SEQ ID NO: 44).

In another aspect, disclosed herein are pharmaceutical compositions comprising a specific binding molecule having the property of binding SLYNTVATL (SEQ ID NO: 1) complexed with HLA-A x 02 and comprising a TCR a chain variable domain and a TCR β chain variable domain, wherein the a chain variable domain amino acid sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:

(a)

(b)

Or (b)

(c)

Optionally with an N-terminal methionine (SEQ ID NOS: 41-43), and

The β chain variable domain comprises the following amino acid sequence:

optionally with an N-terminal methionine (SEQ ID NO: 44).

In some embodiments, the pharmaceutical composition comprises a TCR α chain variable domain and a TCR β chain variable domain, wherein the α chain variable domain has an amino acid sequence selected from the group consisting of:

(a)

(b)

Or (b)

(c)

Optionally with an N-terminal methionine (SEQ ID NOS: 41-43), and

The β chain variable domain has the following amino acid sequence:

optionally with an N-terminal methionine (SEQ ID NO: 44).

Also disclosed herein is a method of treating HIV infection or AIDS in a human subject comprising administering a therapeutically effective amount of a specific binding molecule having the property of binding SLYNTVATL (SEQ ID NO: 1) complexed with HLA-A x 02 and comprising a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the alpha chain variable domain comprises an amino acid sequence selected from the group consisting of:

(a)

(b)

Or (b)

(c)

Optionally with an N-terminal methionine (SEQ ID NOS: 41-43), and

The β chain variable domain comprises the following amino acid sequence:

optionally with an N-terminal methionine (SEQ ID NO: 44).

In some embodiments, the method comprises administering a therapeutically effective amount of a specific binding molecule having the property of binding SLYNTVATL (SEQ ID NO: 1) complexed with HLA-A x 02 and comprising a TCR a chain variable domain and a TCR β chain variable domain, wherein the a chain variable domain has an amino acid sequence selected from the group consisting of:

(a)

(b)

Or (b)

(c)

Optionally with an N-terminal methionine (SEQ ID NOS: 41-43), and

The β chain variable domain has the following amino acid sequence:

optionally with an N-terminal methionine (SEQ ID NO: 44).

In another aspect, disclosed herein is a method comprising: (a) A TCR expression vector comprising a nucleic acid disclosed herein in a single open reading frame, or two different open reading frames encoding the alpha chain and the beta chain, respectively; or (b) a first expression vector comprising a nucleic acid encoding an alpha chain of a TCR disclosed herein, and a second expression vector comprising a nucleic acid encoding a beta chain of a TCR disclosed herein.

In yet another aspect, disclosed herein are isolated or non-naturally occurring cells, particularly T cells that present the TCRs disclosed herein.

Background

Human Immunodeficiency Virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). The virus is an enveloped retrovirus and belongs to the lentivirus group. It is estimated that 4000 tens of thousands of adults and children are infected with HIV (AIDS by the numbers. UNAIDS.2020; accessed June 29,2021, 29 th month, effective https:// www.unaids.org). Current therapies rely on the use of combination antiretroviral therapy (ART) to control viral infections. However, while these treatments are effective, they do not completely eradicate the infection because the viral genes are stably integrated into the host cell chromosome, resulting in the rapid establishment of a long-lived and latently infected CD4+ T cell pool (Siliciano et al, 2003Nat Med,9,727). Viral rebound usually occurs after cessation of treatment, which means that lifelong treatment is required. Most HIV-infected individuals respond strongly to HIV Gag protein during infection, but these T cells fail to eliminate long-lived cd4+ T cells carrying replication competent provirus. To date, the immunotherapeutic strategies tested have not produced significant clinical benefits in controlling and/or reducing the HIV viral pool after treatment (Ward AR et al, semin immunol.2020:101412; barr L et al, journal of viral delivery.2020; 6:100010). Thus, there is a need for new therapeutic approaches that make it possible to eradicate the viral pool and achieve a functional cure.

A novel immunotherapeutic approach involves the use of engineered T Cell Receptors (TCRs) to generate a potent immune response against HIV-infected cells. In nature, T cells and TCRs are generally weak in affinity for antigens, ranging from low micromolar to nanomolar. Engineering the TCR by mutating the antigen recognition site can increase antigen affinity, resulting in an enhancement of the immune response in vivo. In the context of HIV, the enhanced response should be sufficient to destroy replication competent viruses from a low level antigen expressing viral pool. Such engineered TCRs are useful in cell therapy applications using genetically modified T cells (see Vonderheide and June,2014,Immunol Rev,257,7-13). Alternatively, the engineered TCR can be produced as a soluble agent for delivery of cytotoxic or immunostimulating agents to infected cells (Lissin et al ,(2013)."High-Affinity Monocloncal T-cell receptor(mTCR)Fusions.Fusion Protein Technologies for Biophamaceuticals:Applications and Challenges".S.R.Schmidt,Wiley;Boulter, (2003), protein Eng 16 (9): 707-711; liddy et al, (2012), nat Med 8:980-987; WO 03/020763). Similar approaches have been successfully developed for the treatment of certain cancers (Nathan, P et al, new Engl J Med 385,1196-1206 (2021)).

For soluble TCRs to be used as therapeutic agents, it is desirable that their affinity for antigen (K _D) and/or binding half-life be particularly high, for example K _D in the picomolar range and/or a binding half-life of a few hours. This high affinity is necessary to drive a strong response against target cells that present low levels of antigen. In all applications involving affinity engineered TCRs, it is critical that TCRs not only have a higher affinity for antigen than the corresponding wild-type TCRs, but also retain a high level of antigen specificity. In this case, loss of specificity may lead to off-target effects when such TCRs are administered to a patient.

Affinity maturation typically requires the skilled artisan to recognize specific mutations and/or combinations of mutations, including but not limited to substitutions, insertions and/or deletions that may be made to the WT TCR sequence to increase antigen recognition intensity. Methods for identifying specific TCR mutations conferring enhanced affinity are known in the art, for example using a display library (Li et al, (2005) Nat Biotechnol.23 (3): 349-354; holler et al, (2000): proc NATL ACAD SCI U S A;97 (10): 5387-5392). However, in order to significantly increase the affinity of a particular TCR for a particular target, the skilled person needs to select a particular mutation and/or combination of mutations from a large number of possible alternatives. In most cases, the desired affinity and specificity may not be achieved. Mutations required for high affinity and high specificity should also produce TCRs that can be expressed, refolded and purified in reasonable yields and are highly stable in purified form.

Peptide sequence SLYNTVATL (SEQ ID NO: 1) was derived from the p17 gene product of the Gag gene, one of the nine genes that make up the HIV virus, and T cell responses have proven to be particularly effective in controlling viral load, suggesting that this epitope is immunodominant (Rolland et al, 2008,PLoS One,3:e1424;Streeck H et al, J virol.2009;83 (15): 7641-7648; pereyera et al, J virol.2014;88 (22): 12937-12948). The peptide (referred to herein as Gag) is presented by HLA-A.times.02 on the surface of HIV-infected cells. Gag-specific T cells were also detected in individuals receiving ART treatment (viremia free) (Gray CM et al, J immunol.1999;162 (3): 1780-1788; ogg GS et al, J Virol.1999;73 (1): 797-800; seth A et al, J effect Dis.2001;183 (5): 722-729) despite the lower frequency. Thus, gag-HLA-A x 02 complex provides an ideal target for TCR-based identification of HIV-infected cells.

WO2017163064 discloses TCRs that have been mutated relative to WT TCRs that recognize Gag-HLA-A-02 complexes. Cd8+ cytotoxic T cells transduced with the affinity-enhanced TCRs are capable of controlling HIV infection in vitro at an effector target ratio suitable for T cell therapy. These TCRs are capable of recognizing all of the most common viral escape peptides (Varela-Rohena et al, 2008, nat Med,14 (12): 1390-5). Such TCRs are useful in adoptive T cell therapies and soluble TCR-based therapies. Furthermore, in vivo studies described by Yang et al indicate that bispecific molecules incorporating the TCR disclosed in WO2017163064 can treat HIV-infected CD4+ T cells in an individual by redirecting polyclonal (non-HIV-specific) CD8+ T cells to eliminate ART, bypassing HIV-specific immune effectors that may become dysfunctional (Yang H et al, mol Ther.2016;24 (11): 1913-1925).

The inventors have found that certain mutations of the TCR disclosed in WO2017163064 can unexpectedly increase yield and/or stability during escherichia coli production without affecting target binding. Such molecules have desirable properties for clinical development.

Drawings

Fig. 1a shows the amino acid sequences of the TCR alpha and beta chain variable regions of the TCR disclosed in WO2017163064, and fig. 1b shows the amino acid sequences of the bispecific proteins disclosed in WO 2017163064.

FIG. 2 shows the amino acid sequence of the TCR alpha chain variable region of the specific binding molecule of the invention, wherein the F50K mutation is highlighted in gray.

FIG. 3 is a graph showing the yield of bispecific protein with different mutations per culture volume.

FIG. 4 shows a graph of binding kinetics for M49K and F50K mutants.

FIG. 5 shows the amino acid sequence of the TCR alpha chain variable region of the specific binding molecule of the invention, wherein the F50K and S96A mutations are highlighted in grey.

FIG. 6 shows the amino acid sequence of the bispecific proteins of the invention.

Figure 7 shows a graph of the efficacy of T cell redirection as determined by ifnγ (upper) and GrB (lower) release.

Detailed Description

In a first aspect, the present invention provides a specific binding molecule having the property of binding SLYNTVATL (SEQ ID NO: 1) complexed with HLA-A.02 and comprising a TCR alpha chain variable domain and a TCR beta chain variable domain,

Wherein the alpha chain variable domain comprises an amino acid sequence selected from the group consisting of:

a)

AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNSGYALN FGKGTSLLVT P(SEQ ID NO:2);

b)

AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMFL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P(SEQ ID NO:3);

Or c)

AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGR FTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P(SEQ ID NO:4),

Optionally with an N-terminal methionine (SEQ ID NOS: 41-43), and

The β chain variable domain comprises the following amino acid sequence:

DAGVTQSPTH LIKTRGQQVT LRCSPKSGHD TVSWYQQALG QGPQFIFQAV RGVERQRGNF PDRFSGHQFP NYSSELNVNA LLLGDSALYL CASSDTVSYE QYFGPGTRLT VT(SEQ ID NO:5),

optionally with an N-terminal methionine (SEQ ID NO: 44).

In the above amino acid sequences of the TCR a and TCR β chain variable domains of the specific binding molecules of the invention, the shaded residues represent CDRs and the underlined residues represent mutations relative to the TCR a and TCR β chain variable domains of the TCRs disclosed in WO2017163064 (see figures 1a and 1b, respectively). When amino acids are expressed herein in numerical positions, the numbering of that position assumes the presence of an optional N-terminal methionine.

The present invention provides specific binding molecules that can be produced in increased yields and/or that have unexpectedly high stability, while also unexpectedly retaining the advantageous properties of the specific binding molecules from which they were derived. These properties include good antigen binding properties (including picomolar antigen affinity), long binding half-life, ability to mediate potent immune activation against HIV-infected cells presenting very low levels of antigen when fused to the activating moiety, and high levels of specificity. The specific binding molecules of the invention are particularly suitable for use as soluble targeting agents for the treatment of HIV-infected individuals.

Specific binding molecules or binding fragments thereof can be used to produce molecules with desirable therapeutic properties, such as a supraphysiological affinity for the target, a long binding half-life, high specificity for the target, and good stability. The invention also includes bispecific, or bifunctional or fusion molecules incorporating specific binding molecules or binding fragments thereof and T cell redirecting moieties. Such molecules can mediate potent specific responses against HIV-infected cells by redirecting and activating polyclonal T cell responses. In addition, the use of binding molecules with super-physiological affinity specificity helps to identify and clear HIV-infected cells presenting low levels of peptide-HLA. Or specific binding molecules or binding fragments may be fused with other therapeutic and/or diagnostic agents, and/or integrated into engineered T cells for adoptive therapy.

TCR domain sequences may be defined with reference to IMGT nomenclature, which is well known and readily understood by those skilled in the TCR art. See, for example, ：LeFranc and LeFranc,(2001)."T cell Receptor Factsbook",Academic Press;Lefranc,(2011),Cold Spring Harb Protoc 2011(6):595-603;Lefranc,(2001),Curr Protoc Immunol Appendix 1:Appendix 10; and Lefranc (2003), leukemia 17 (1): 260-266. Briefly, αβ TCRs consist of two disulfide-linked chains. Each chain (α and β) is generally considered to have two domains, a variable domain and a constant domain. The short linking region connects the variable and constant domains and is generally considered to be part of the alpha variable domain. furthermore, the β chain typically comprises a short diversity region beside the linking region, which region is also typically considered to be part of the β variable domain. The variable domain of each chain is located at the N-terminus and comprises three Complementarity Determining Regions (CDRs) embedded in a framework sequence (FR). CDRs contain the recognition sites for peptide-MHC binding. There are several genes encoding the alpha chain variable (V.alpha.) region and several genes encoding the beta chain variable (V.beta.) region, which are distinguished by their framework, CDR1 and CDR2 sequences and partially defined CDR3 sequences. In IMGT nomenclature, the V.alpha.and V.beta.genes are denoted by the prefixes TRAV and TRBV, respectively (Folch and Lefranc, (2000), exp Clin Immunogenet 17 (1): 42-54; Scaviner and Lefranc, (2000), exp Clin Immunogenet 17 (2): 83-96; leFranc and LeFranc, (2001), "T cell Receptor Factsbook", ACADEMIC PRESS). Likewise, there are several junction genes or J genes, called TRAJ or TRBJ, for the alpha and beta chains, respectively, and a diversity gene or D gene, called TRBD (Folch and Lefranc, (2000), exp Clin Immunogenet 17 (2): 107-114, for the beta chain; Scaviner and Lefranc, (2000), exp Clin Immunogenet 17 (2): 97-106; leFranc and LeFranc, (2001), "T cell Receptor Factsbook", ACADEMIC PRESS). The great diversity of T cell receptor chains is caused by the combined rearrangement between the various V, J and D genes, including allelic variants and linkage diversity (Arstila et al, (1999) Science286 (5441): 958-961; robin et al, (2009) Blood 114 (19): 4099-4107) constant domains or C-chains of TCRα and β are referred to as TRAC and TRBC, respectively (Lefranc, (2001), curr Protoc Immunol Appendix: appdix 10).

As used herein, the term "specific binding molecule" refers to a molecule capable of specifically binding to a target antigen. Such molecules can take a variety of different forms as discussed herein, and can be bispecific, i.e., they can have a first binding region that specifically binds a first target antigen and a second binding region that specifically binds a second target antigen. Furthermore, fragments of the specific binding molecules of the invention are also contemplated. Fragment refers to a portion of a specific binding molecule that retains specific binding to a target antigen.

The term "mutation" encompasses substitutions, insertions and deletions. Mutations to native (also known as parent, native, unmutated, wild-type or scaffold) specific binding molecules may confer beneficial therapeutic properties, such as high affinity, high specificity and high potency; for example, mutations may include those that enhance the binding affinity (K _D) and/or the binding half-life (t 1/2) of the specific binding molecule to the SLYNTVATL (SEQ ID NO: 1) -HLA-A.02 complex. In the present invention, mutations may additionally increase yield and/or stability while unexpectedly retaining the beneficial properties described above.

The alpha chain variable domain comprises the amino acid sequence:

a)

The inventors found that mutating the F residue at position 50 to K can increase yield during e.coli production, but unexpectedly this did not affect target binding.

Or the alpha chain variable domain comprises the amino acid sequence:

b)

The inventors found that mutating the S residue at position 96 to a improved stability, but again unexpectedly, target binding was not affected.

Preferably, the alpha chain variable domain comprises the amino acid sequence:

c)

specific binding members comprising such a chain variable domains unexpectedly increase both yield and stability, and target binding is unaffected.

The specific binding molecules of the invention are suitable for high yield purification, particularly in soluble form, and particularly when expressed in E.coli. The yield can be determined from the amount of correctly folded material obtained at the end of the purification process relative to the original culture volume. Optionally, the yield is as determined in example 1 herein. High yields generally mean yields of greater than 2mg/L, or more preferably greater than 3mg/L, or greater than 4mg/L or greater than 5mg/L, or higher.

The specific binding molecules of the invention may have improved stability, particularly in purified form. Stability in this context generally means that accumulation of decomposition products and/or non-uniformity of the purified material increases over time. In general, accelerated stability studies can be performed to provide an indication of long-term molecular stability; in this case, the purification material may be exposed to pressure conditions, such as extreme temperatures or pH. For example, accumulation of acidic species at high pH conditions can be used as a measure of molecular instability. This procedure is further described in example 2. The relative decrease in the main peak and the corresponding increase in acidic species is preferably less than 35%, less than 30%, less than 25%, less than 20%, and more preferably less than 15% when measured under the conditions described in example 2. In addition, stability can alternatively be assessed by measuring the relative abundance of amino acid modifications (e.g., deamidation) over time. Preferably, the change in relative abundance is less than 15%, less than 10%, less than 7%, less than 5%, less than 3%, and more preferably less than 1%. Minimizing the risk of molecular instability is critical to successful clinical development.

In addition to the mutations described above, the specific binding molecules of the invention may have one or more additional mutations in their alpha chain variable domains. These mutations may be selected from A2Q, V, 73, I, Q K and P82L.

The combination of these mutations can be as follows:

A2Q；

V73I；

Q81K；

P82L；

a2Q and V73I;

a2Q and Q81K;

a2Q and P82L;

V73I and Q81K;

V73I and P82L;

Q81K and P82L;

a2Q, V73I and Q81K;

a2Q, V73I and P82L;

V73I, Q K and P82L; or (b)

A2Q, V73I, Q K and P82L.

These mutations may occur in any of SEQ ID NOs 2,3, 4, 41, 42 and 43.

Within the scope of the present application are phenotypically silent variants of any specific binding molecule disclosed herein. As used herein, the term "phenotypically silent variant" should be understood to refer to a specific binding molecule comprising one or more other amino acid changes (including substitutions, insertions and deletions) in addition to the above-described changes, which specific binding molecule has a phenotype similar to the corresponding specific binding molecule without the changes. For the purposes of the present application, specific binding molecule phenotypes include antigen binding affinity (K _D and/or binding half-life), antigen specificity, yield and stability. The phenotype silencing variant pair SLYNTVATL (SEQ ID NO: 1) HLA-A x 02 complex has a measured K _D and/or binding half-life of less than 50%, or more preferably less than 20%, of the K _D and/or binding half-life of the corresponding specific binding molecule that does not comprise the change, when measured under the same conditions (e.g. at 25 ℃ and on the same SPR chip). Suitable conditions are further provided in example 3 in WO 2017163064. Yield and stability are further defined above. Antigen specificity is further defined below. As known to those skilled in the art, it is possible to generate specific binding molecules whose variable domains are altered compared to those detailed above, but without altering their affinity for interaction with the SLYNTVATL (SEQ ID NO: 1) HLA-A.02 complex. In particular, such silent mutations may incorporate portions of sequences known not to directly participate in antigen binding (e.g., framework regions, or CDR portions that do not contact peptide antigens). Such simple variants are included within the scope of the application.

Phenotype silencing variants may be produced by introducing one or more conservative substitutions and/or one or more tolerable substitutions. Tolerable substitutions are those that do not fall within the conservative definition provided below, but still appear phenotypically silent. Conservative substitutions refer to the substitution of one or more amino acids with substituted amino acids having similar properties. Those skilled in the art know that various amino acids have similar properties and are therefore "conserved". One or more such amino acids of a protein, polypeptide or peptide may typically be substituted with one or more other such amino acids without eliminating the desired activity of the protein, polypeptide or peptide. Thus, the amino acids glycine, alanine, valine, leucine and isoleucine can generally be substituted for each other (amino acids having aliphatic side chains). Among these possible substitutions, glycine and alanine are preferably used instead of each other (because they have relatively short side chains), and valine, leucine and isoleucine are preferably used instead of each other (because they have large hydrophobic aliphatic side chains). Other amino acids that may be substituted for each other in general include: phenylalanine, tyrosine, and tryptophan (amino acids having aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine and methionine (amino acids with sulfur-containing side chains). It is understood that amino acid substitutions may be made using naturally occurring or non-naturally occurring amino acids within the scope of the invention. For example, it is contemplated herein that the methyl group on alanine may be substituted with ethyl groups, and/or that minor changes may be made to the peptide backbone. Whether natural or synthetic amino acids are used, preferably only L-amino acids are present.

Mutations (which include conservative and tolerable substitutions, insertions, and deletions) may be introduced into the provided sequences using any suitable method, including, but not limited to: those methods based on Polymerase Chain Reaction (PCR), restriction enzyme-based cloning or Ligation Independent Cloning (LIC) procedures. These methods are described in detail in many standard molecular biology texts. For more details on Polymerase Chain Reaction (PCR) and restriction enzyme based cloning see Sambrook & Russell, (2001) Molecular Cloning-A Laboratory Manual (3 rd Ed.) CSHL PRESS. For more information on Ligation Independent Cloning (LIC) procedures, see Rashtchian, (1995) Curr Opin Biotechnol (1): 30-6. The specific binding molecule sequences provided herein may be obtained by solid state synthesis or any other suitable method known in the art.

The specific binding molecules of the invention have the property of binding SLYNTVATL-HLA-A x 02 complex ("SLYNTVATL" is disclosed as SEQ ID NO: 1). The specific binding molecules of the invention exhibit a high degree of specificity for SLYNTVATL-HLA-A-02 complex ("SLYNTVATL" is disclosed as SEQ ID NO: 1) and are therefore particularly suitable for therapeutic use. The specific binding molecules of the present invention are specifically directed to their ability to recognize antigen-positive target cells while having minimal ability to recognize antigen-negative target cells. Antigen positive cells are those cells that have been determined to be infected with HIV and/or those cells that have been determined to present SLYNTVATL-HLA-A-02 complex ("SLYNTVATL" is disclosed as SEQ ID NO: 1), or escape variants of SLYNTVATL (SEQ ID NO: 1) presented by HLA-A-02 as discussed herein. The specific binding molecules of the invention may bind to a target peptide complex when bound to one or more of the HLA-A x 02 subtypes, e.g., the specific binding molecules of the invention may bind to a target peptide complex when bound to HLA-A x 02:01, and/or the specific binding molecules of the invention may bind to a target peptide complex when bound to HLA-A x 02:05 and/or HLA-A x 02:06 and/or HLA-A x 02:07 and/or HLA-A x 02:02.

Specificity may be measured in vitro, for example in a cellular assay such as those described in example 5 of WO 2017163064. To test for specificity, the specific binding molecules may be in soluble form and associated with immune effectors, and/or may be expressed on the surface of cells (e.g., T cells). Specificity can be determined by measuring the level of T cell activation in the presence of antigen positive and antigen negative target cells as defined above. The minimum recognition of antigen-negative target cells is defined as a level of T cell activation of less than 20%, preferably less than 10%, preferably less than 5%, more preferably less than 1% of the level produced in the presence of antigen-positive target cells, when measured under the same conditions and at the concentration of the therapeutically relevant specific binding molecule. For soluble TCRs associated with immune effectors, a therapeutically relevant concentration may be defined as a concentration of 10 ^-9 M or less, and/or a concentration up to 100-fold, preferably up to 1000-fold higher than the corresponding EC50 or IC50 value. Preferably, for soluble specific binding molecules associated with immune effectors, there is at least a 100-fold, at least a 1000-fold, at least a 10000-fold difference between the EC50 or IC50 value of T cell activation against antigen positive cells relative to T cell activation against antigen negative cells-this difference may be referred to as a therapeutic window. Additionally or alternatively, the therapeutic window may be calculated based on the lowest effective concentration ("LOEL") observed for normal cells and HIV-infected cells. Antigen positive cells can be obtained by peptide pulsing, low levels of antigen presentation comparable to those of latently infected cells can be obtained using appropriate peptide concentrations (e.g., 10 ^-9 M peptides such as Bossi et al, (2013) Oncominol. 1;2 (11): e 26840), or they can present the peptides naturally. Preferably, both the antigen positive cells and the antigen negative cells are human cells. Preferably, the antigen positive cells are human cells, such as HIV-infected cd4+ T cells. Antigen negative cells preferably include cells derived from healthy human tissue, or non-HIV infected cd4+ T cells.

Specificity may additionally or alternatively relate to the ability of a specific binding molecule to bind to SLYNTVATL-HLA-A-02 complex ("SLYNTVATL" is disclosed as SEQ ID NO: 1) and not to a surrogate peptide-HLa complex set. Preferably, the surrogate peptide-HLA complex comprises HLA-a x 02. This can be determined, for example, by the Biacore method of example 3 of WO 2017163064. The panel may comprise at least 5, and preferably at least 10 surrogate peptide-HLA complexes. The replacement peptide may share a low level of sequence identity with SLYNTVATL (SEQ ID NO: 1) and may be presented naturally. The surrogate peptide is preferably derived from a protein expressed in healthy human tissue (i.e., not HIV-infected cells). The binding of the specific binding molecule to SLYNTVATL-HLA-A-02 complex ("SLYNTVATL" is disclosed as SEQ ID No. 1) may be at least 2-fold, more preferably at least 10-fold, or at least 100-fold, or at least 1000-fold, or at least 3000-fold higher than other naturally presented peptide HLa complexes.

An alternative or additional method of determining the specificity of a specific binding molecule may be to identify the peptide recognition motif of the specific binding molecule using sequential mutagenesis (e.g., alanine scanning) of the target peptide. Residues forming part of the binding motif are those residues that do not allow substitution. Disallowed substitutions may be defined as those peptide positions where the binding affinity of the specific binding molecule is reduced by at least 50% or at least 80% relative to the binding affinity of the unmutated peptide. This method is further described in camelon et al, (2013), SCI TRANSL med.2013aug 7;5 (197) in 197ra103 and WO 2014096803. It will be appreciated that this approach may also be applied to specific binding molecules of the invention. In this case, the specificity of the specific binding molecule can be determined by identifying peptides containing alternative motifs (particularly peptides containing alternative motifs in the human proteome) and testing the binding of these peptides to the specific binding molecule. Binding of the specific binding molecule to one or more surrogate peptides may indicate lack of specificity. In this case, it may be necessary to further test the specificity of the specific binding molecule by a cellular assay. The low tolerance of the substitution (alanine) in the central part of the peptide means that the specific binding molecule has a high specificity and thus shows a low risk of cross-reacting with the replacement peptide.

The specific binding molecules of the invention may also bind complexes containing the natural escape variant of SLYNTVATL (SEQ ID NO: 1) presented by HLA-A-02. Escape variants of peptide SLYNTVATL (SEQ ID NO: 1) have been isolated from AIDS patients and include the following variants (Sewell et al, (1997) Eur J immunol.27:2323-2329):

SLFNTVATL(SEQ ID NO:6)

SLFNTVAVL(SEQ ID NO:7)

SLSNTVATL(SEQ ID NO:8)

SSFNTVATL(SEQ ID NO:9)

SLLNTVATL(SEQ ID NO:10)

SLYNTIATL(SEQ ID NO:11)

SLYNTIAVL(SEQ ID NO:12)

SLFNTIATL(SEQ ID NO:13)

SLFNTIAVL(SEQ ID NO:14)

SLYNFVAVL(SEQ ID NO:15)。

The specific binding molecules of the invention have desirable safety profiles for use as therapeutic agents. In this case, the specific binding molecule may be in a soluble form and is preferably fused to an immune effector. Suitable immune effectors include, but are not limited to, cytokines, such as IL-2 and IFN-gamma; superantigens and mutants thereof; chemokines, such as IL-8, platelet factor 4, melanoma growth stimulatory proteins; antibodies and antibody-like scaffolds, including fragments, derivatives, and variants thereof (e.g., anti-CD 3, anti-CD 28, or anti-CD 16) that bind to an antigen on an immune cell (e.g., a T cell or NK cell); fc receptors or complement activators. The ideal safety means that in addition to exhibiting good specificity, the specific binding molecules of the invention may have passed further preclinical safety tests. Examples of such assays include whole blood assays to determine that cytokine release is minimal in the presence of whole blood and therefore there is less risk of causing potential cytokine release syndrome in vivo, and allo-reactivity tests to determine that it is less likely that other HLA types are identified.

The specific binding molecule pair SLYNTVATL-HLA-A-02 complex of the invention ("SLYNTVATL" is disclosed as SEQ ID NO: 1) preferably has a K _D of less than 100nM, for example about 50nM to about 1pM, and/or a binding half-life (T1/2) to the complex in the range of about 1 minute to about 50 hours or more. Certain specific binding molecules of the invention have a K _D of about 1pM to about 1nM, about 1pM to about 500pM, about 1pM to about 300pM for the complex. Certain TCRs of the present invention have a K _D of about 50pM to about 200pM for the complex. The specific binding molecules of the invention have a binding half-life (T1/2) for the complex in the range of about 1 minute to about 50 hours or more (e.g., 100 hours), about 30 minutes to about 50 hours or more (e.g., 100 hours), or about 6 hours to about 50 hours or more (e.g., 100 hours). When coupled to a detectable label or therapeutic agent, all such specific binding molecules are well suited for use as therapeutic and/or diagnostic agents. Certain specific binding molecules of the invention may be suitable for adoptive therapeutic applications, such specific binding molecules having a K _D for the complex of about 50nM to about 200nM, and/or a binding half-life for the complex of about 3 seconds to about 12 minutes.

Methods for determining binding affinity (inversely proportional to equilibrium constant K _D) and binding half-life (expressed as T1/2) are known to those skilled in the art. In a preferred embodiment, the binding affinity and binding half-life are determined using Surface Plasmon Resonance (SPR) or Biological Layer Interferometry (BLI), respectively, for example using a BIAcore device or an Octet device, respectively. A preferred method is provided in example 3 of WO 2017163064. It will be appreciated that doubling the affinity of the specific binding molecule results in halving of K _D. T1/2 is calculated by dividing ln2 by the dissociation rate (k _off). Thus, doubling T1/2 would result in halving k _off. The K _D and K _off values of TCRs are typically measured for soluble forms of TCRs, i.e., forms truncated to remove cytoplasmic and transmembrane domain residues (including single chain TCRs and/or TCRs incorporating non-native disulfide bonds or other dimerization domains). To account for differences between independent measurements, particularly for interactions with dissociation times exceeding 20 hours, the binding affinity and/or binding half-life of a given specific binding molecule may be measured multiple times (e.g., 3 times or more) using the same assay protocol, and the results averaged. In order to compare binding data between two samples (i.e. two different specific binding molecules and/or two formulations of the same specific binding molecule), it is preferred to use the same assay conditions (e.g. temperature) for measurements, such as those described in example 3 of WO 2017163064.

Certain preferred mutated specific binding molecules of the invention are capable of producing in vitro a high efficiency T cell response against antigen positive cells, particularly those cells typical of HIV-infected CD4 cells that present low levels of antigen (i.e., on the order of 5-100), which are typical HIV-infected CD4 cells. Such specific binding molecules may be in soluble form and linked to immune effectors (e.g., anti-CD 3 antibodies). The T cell response measured may be the release of a T cell activation marker (e.g., interferon gamma or granzyme B), or target cell killing, or other measure of T cell activation, such as T cell proliferation. Preferably, a high efficiency reaction is one having an EC50 or IC50 value in the pM range, e.g. 100pM or less, preferably 50pM or less, e.g. between 50pM and 1 pM.

The specific binding molecules of the invention may comprise a TCR variable domain. Preferably, the TCR variable domain comprises a heterodimer of an alpha chain and a beta chain.

In the specific binding molecules of the invention, the variable domains and the constant domains present and/or any other domains may be organized in any suitable form/arrangement. Examples of such arrangements are well known in the antibody arts. The skilled artisan knows the similarity between antibodies and TCRs, and such an arrangement can be applied to TCR variable and constant domains (Brinkman et al, MAbs.2017Feb-Mar;9 (2): 182-212). For example, the variable domains may be arranged in a monoclonal TCR format, wherein the two chains are linked by disulfide bonds, whether within a constant domain or a variable domain, or the variable domain is fused to one or more dimerization domains. Or the variable domains may be arranged in single chain form with or without the presence of one or more constant domains, or the variable domains may be arranged in diabody form.

The specific binding molecules of the invention may comprise at least one TCR constant domain or fragment thereof, e.g. an alpha chain TRAC constant domain and/or a beta chain TRBC1 or TRBC2 constant domain. It will be appreciated by those skilled in the art that the terms TRAC and TRBC1/2 also encompass natural polymorphic variants, e.g., a change of N to K at position 4 of TRAC (Bragado et al, international immunology 1994Feb;6 (2): 223-30).

One or both of the constant domains, if present, may comprise mutations, substitutions or deletions relative to the native constant domain sequence. The constant domain may be truncated, i.e., not have a transmembrane domain or cytoplasmic domain. Alternatively, the constant domain may be full length, meaning that extracellular, transmembrane and cytoplasmic domains are all present. The TRAC and TRBC domain sequences can be modified by truncation or substitution to delete the native disulfide bond between Cys4 of TRAC exon 2 and Cys2 of TRBC1 or TRBC2 exon 2. The alpha and/or beta chain constant domain sequences may introduce disulfide bonds between residues of the respective constant domains, for example as described in WO 03/020763. Preferably, the alpha and beta chain constant domain amino acid sequences are modified by substitution of Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2 with cysteine residues that form a non-native disulfide bond between the TCR alpha and beta constant domains. TRBC1 or TRBC2 may further comprise a cysteine to alanine mutation at position 75 of the constant domain and an asparagine to aspartic acid mutation at position 89 of the constant domain. One or both of the extracellular constant domains present in the αβ heterodimers of the present invention may be further truncated at the C-terminus or C-terminus, e.g., by up to 15, up to 10, or up to 8 or less amino acids. One or both of the extracellular constant domains present in the αβ heterodimers of the present invention may be truncated at the C-terminus or C-terminus, e.g., up to 15, or up to 10, or up to 8 amino acids. The C-terminus of the extracellular constant domain of the alpha chain may be truncated by 8 amino acids.

Or there may be no TCR constant domain other than a full length or truncated constant domain. Thus, the specific binding molecules of the invention may consist of the variable domains of the TCR alpha and beta chains, optionally with other domains as described herein. Other domains include, but are not limited to, immune effector domains (e.g., antibody domains), fc domains or albumin binding domains, therapeutic agents, or detectable labels.

Single chain forms include, but are not limited to, αβ TCR polypeptides of the type V.alpha. -L-V.beta., V.beta. -L-V.alpha., V.alpha. -C.alpha. -L-V.beta., V.alpha. -L-V.beta. -C.beta., V.alpha. -C.alpha. -L-V.beta. -C.alpha., wherein V.alpha and V.beta. Are TCR alpha and beta variable domains, respectively, C.alpha and C.beta. Are TCR alpha and beta constant domains, respectively, and L is a linker sequence (Weidanz et al, (1998) J Immunol methods Dec 1;221 (1-2): 59-76; epel et al, (2002), cancer Immunol. Nov; 51 565-73; WO 2004/033685; WO 9918129). Linker sequences are typically flexible in that they consist essentially of amino acids, such as glycine, alanine, and serine, which do not have bulky side chains that may limit flexibility. Or a more rigid joint may be desirable. The available length or optimal length of the linker sequence can be easily determined. The linker sequence is typically less than about 12 amino acids in length, for example less than 10 amino acids, or 2-10 amino acids. The length of the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. Examples of suitable linkers that may be used in the multidomain binding molecules of the invention include, but are not limited to ：GGGGS(SEQ ID NO:16)、GGGSG(SEQ ID NO:17)、GGSGG(SEQ ID NO:18)、GSGGG(SEQ ID NO:19)、GSGGGP(SEQ ID NO:20)、GGEPS(SEQ ID NO:21)、GGEGGGP(SEQ ID NO:22) and GGEGGGSEGGGS (SEQ ID NO: 23) (as described in WO 2010/133828) and GGGSGGGG (SEQ ID NO: 24). Other linkers may include sequences having one or more of the following sequence motifs: GGGS (SEQ ID NO: 25), GGGGS (SEQ ID NO: 26), TVLRT (SEQ ID NO: 27), TVSSAS (SEQ ID NO: 28) and TVLSSAS (SEQ ID NO: 29). If present, one or both of the constant domains may be full length, or they may be truncated and/or contain mutations as described above. Preferably, the single chain TCR is soluble. In certain embodiments, single chain TCRs of the invention may incorporate disulfide bonds between residues of the respective constant domains, as described in WO 2004/033685. Single chain TCRs are further described in WO2004/033685; WO98/39482; WO01/62908; weidanz et al, (1998) J Immunol Methods221 (1-2): 59-76; Hoo et al, (1992) Proc NATL ACAD SCI U S A89 (10): 4759-4763; schodin (1996) Mol Immunol 33 (9): 819-829.

The TCR variable domains may be arranged in diabody form. In diabody format, two single chain fragments dimerize in the head-to-tail direction to form a compact molecule with a molecular weight similar to tandem scFv (-50 kDa).

The invention also includes particles displaying the specific binding molecules of the invention, and the particles are separately contained in a library of particles. Such particles include, but are not limited to, phage, yeast cells, ribosomes, or mammalian cells. Methods of producing such particles and libraries are known in the art (see, e.g., WO2004/044004; WO01/48145, chervin et al, (2008) J.Immuno. Methods 339.2:175-184).

The specific binding molecules of the invention can be used to deliver detectable labels or therapeutic agents to antigen presenting cells and tissues containing antigen presenting cells. Thus, they can be conjugated to a detection label (for diagnostic purposes, wherein specific binding molecules are used, for example, to detect the presence of cells presenting cognate antigen); and/or therapeutic agents, including immune effectors; and/or association (covalently or otherwise) of Pharmacokinetic (PK) modifying moieties.

Examples of PK modifying moieties include, but are not limited to, PEG (Dozier et al, (2015) Int JMol Sci.Oct 28;16 (10): 25831-64 and Jevseva et al, (2010) Biotechnol J.Jan;5 (1): 113-28), PASylation (SCHLAPSCHY et al, (2013) Protein end Des Sel.Aug;26 (8): 489-501), albumin and albumin binding domains (Dennis et al, (2002) J Biol chem.Sep 20;277 (38): 35035-43) and/or unstructured polypeptides (Schellenberger et al, (2009) Nat Biotechnol.Dec;27 (12): 1186-90). Additional PK modifying moieties include antibody Fc fragments. PK modifying moieties can be used to extend the in vivo half-life of the specific binding molecules of the invention.

When an immunoglobulin Fc domain is used, it may be any antibody Fc region. The Fc region is the tail region of an antibody, which interacts with cell surface Fc receptors and some proteins of the complement system. The Fc region typically comprises two polypeptide chains, each having two or three heavy chain constant domains (known as CH2, CH3 and CH 4) and one hinge region. The two chains are linked by disulfide bonds within the hinge region. The Fc domains of the immunoglobulin subclasses IgG1, igG2 and IgG4 bind to and undergo FcRn-mediated recycling, providing a longer circulation half-life (3-4 weeks). The interaction of IgG with FcRn is located in the Fc region, covering portions of the CH2 and CH3 domains. Preferred immunoglobulin fcs for use in the present invention include, but are not limited to, fc domains from IgG1 or IgG 4. Preferably the Fc domain is derived from a human sequence. The Fc region may also preferably contain KiH mutations that promote dimerization, as well as mutations that prevent interaction with activating receptors (i.e., functional silencing molecules). The immunoglobulin Fc domain may be fused to the C or N terminus of the other domain (i.e., the TCR variable domain and/or the TCR constant domain and/or the immune effector domain) in any suitable order or configuration. Immunoglobulin Fc may be fused to one or more other domains (i.e., TCR variable domains and/or TCR constant domains and/or immune effector domains) via a linker. Linker sequences are generally flexible in that they are composed primarily of amino acids (e.g., glycine, alanine, and serine) that do not have bulky side chains that may limit flexibility. Or a more rigid joint may be desirable. The available length or optimal length of the linker sequence can be easily determined. The linker sequence is typically less than about 12 amino acids in length, for example less than 10 amino acids, or 2-10 amino acids. The length of the linker may be 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. Examples of suitable linkers that may be used in the multidomain binding molecules of the invention include, but are not limited to ：GGGGS(SEQ ID NO:16)、GGGSG(SEQ ID NO:17)、GGSGG(SEQ ID NO:18)、GSGGG(SEQ ID NO:19)、GSGGGP(SEQ ID NO:20)、GGEPS(SEQ ID NO:21)、GGEGGGP(SEQ ID NO:22) and GGEGGGSEGGGS (SEQ ID NO: 23) (as described in WO 2010/133828) and GGGSGGGG (SEQ ID NO: 24). Other linkers may include sequences having one or more of the following sequence motifs: GGGS (SEQ ID NO: 25), GGGGS (SEQ ID NO: 26), TVLRT (SEQ ID NO: 27), TVSSAS (SEQ ID NO: 28) and TVLSSAS (SEQ ID NO: 29). When an immunoglobulin Fc is fused to a TCR, it may be fused to an alpha chain or a beta chain, with or without a linker. Furthermore, individual chains of Fc may be fused to individual chains of TCR.

Preferably, the Fc region may be derived from the IgG1 or IgG4 subclass. Both chains may comprise all or part of the CH2 and CH3 constant domains and the hinge region. The hinge region may correspond substantially or in part to a hinge region from IgG1, igG2, igG3 or IgG 4. The hinge may comprise all or part of the core hinge domain and all or part of the lower hinge region. Preferably, the hinge region comprises at least one disulfide bond connecting the two chains.

The Fc region may comprise mutations relative to the WT sequence. Mutations include substitutions, insertions and deletions. Such mutations can be used to introduce desired therapeutic properties. For example, to promote heterodimerization, a "knob-in-mortar" (KiH) mutation may be designed in the CH3 domain. In this case, one strand is designed to contain large protruding residues (i.e., knob), e.g., Y, while the other strand is designed to contain a complementary pocket (i.e., hole). Suitable positions for KiH mutations are known in the art. Additionally or alternatively, mutations may be introduced to eliminate or reduce binding to Fcy receptors and/or increase binding to FcRn, and/or prevent Fab arm exchange, or to remove protease sites. Additionally or alternatively, mutations may be made to improve manufacturability, e.g., to remove or alter glycosylation sites.

The PK modifying moiety may also be an albumin binding domain, which may also act to extend half-life. As known in the art, albumin has a long circulation half-life of 19 days, in part because its size is above the renal threshold, and its specific interaction and recycling by FcRn. Attachment to albumin is a well known strategy to improve the in vivo circulation half-life of therapeutic molecules. Albumin may be non-covalently linked by use of specific albumin binding domains, or covalently linked by binding or direct gene fusion. Sleep et al, biochim Biophys acta.2013Dec;1830 An example of a therapeutic molecule that utilizes attachment to albumin to improve half-life is given in (12) 5526-34.

The albumin binding domain may be any moiety capable of binding albumin, including any known albumin binding moiety. Albumin binding domains may be selected from endogenous or exogenous ligands, small organic molecules, fatty acids, peptides and proteins that specifically bind albumin. Examples of preferred albumin binding domains include short peptides, such as Dennis et al, J Biol chem.2002sep20; 277 (38) short peptides described in 35035-43 (e.g., peptide QRLMEDICLPRWGCLWEDDF (SEQ ID NO: 37)); proteins engineered to bind albumin, such as antibodies, antibody fragments, and antibody-like scaffolds, such as those commercially provided by GSK(O' Connor-Semmes et al, clin Pharmacol Ther.2014Dec;96 (6): 704-12) and commercially available from Ablynx(Van Roy et al ARTHRITIS RES Ther.2015May20; 17:135); and proteins based on albumin binding domains found in nature, e.g. streptococcal protein G protein (Stork et al, eng Des sel.2007Nov;20 (11): 569-76), e.g. as supplied by Affibody commerce

Preferably, the albumin is Human Serum Albumin (HSA). The affinity of the albumin binding domain for human albumin may be in the picomolar to micromolar range. Given the extremely high concentration of albumin in human serum (35-50 mg/ml, about 0.6 mM), it is calculated that substantially all albumin binding domains bind albumin in vivo.

The albumin binding moiety may be fused to the C or N terminus of the other domain (i.e., the TCR variable domain and/or the TCR constant domain and/or the immune effector domain) in any suitable order or configuration. The albumin binding moiety may be fused to one or more of the other domains (i.e., TCR variable domain and/or TCR constant domain and/or immune effector domain) via a linker. Linker sequences are generally flexible in that they are composed primarily of amino acids (e.g., glycine, alanine, and serine) that do not have bulky side chains that may limit flexibility. Or a more rigid joint may be desirable. The available length or optimal length of the linker sequence can be easily determined. The linker sequence is typically less than about 12 amino acids in length, for example less than 10 amino acids, or 2-10 amino acids. The length of the linker may be 1, 2, 3,4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. Examples of suitable linkers that may be used in the multidomain binding molecules of the invention include, but are not limited to ：GGGGS(SEQ ID NO:16)、GGGSG(SEQ ID NO:17)、GGSGG(SEQ ID NO:18)、GSGGG(SEQ ID NO:19)、GSGGGP(SEQ ID NO:20)、GGEPS(SEQ ID NO:21)、GGEGGGP(SEQ ID NO:22) and GGEGGGSEGGGS (SEQ ID NO: 23) (as described in WO 2010/133828) and GGGSGGGG (SEQ ID NO: 24). Other linkers may include sequences having one or more of the following sequence motifs: GGGS (SEQ ID NO: 25), GGGGS (SEQ ID NO: 26), TVLRT (SEQ ID NO: 27), TVSSAS (SEQ ID NO: 28) and TVLSSAS (SEQ ID NO: 29). When the albumin binding moiety is linked to a specific binding molecule, it may be linked to an alpha chain or a beta chain, with or without a linker.

Detectable labels for diagnostic purposes include, for example, fluorescent labels, radioactive labels, enzymes, nucleic acid probes, and contrast agents.

For some purposes, the specific binding molecules of the invention may aggregate into a complex comprising a plurality of specific binding molecules to form a multivalent specific binding molecule complex. Many human proteins contain multimerization domains that can be used to produce multivalent specific binding molecule complexes. For example, the tetramerization domain of p53 has been used to produce tetramers of scFv antibody fragments, which exhibit increased serum persistence and significantly reduced dissociation rates compared to monomeric scFv fragments (Willuda et al, (2001) j.biol.chem.276 (17) 14385-14392). Hemoglobin also has tetramerization domains that can be used for such applications. The multivalent specific binding molecule complexes of the invention can have enhanced complex binding capacity compared to the non-multimeric native (also referred to as parent, native, unmutated wild-type or scaffold) T cell receptor heterodimers of the invention. Thus, multivalent complexes of the specific binding molecules of the invention are also encompassed by the invention. Such multivalent specific binding molecule complexes according to the invention are particularly useful for tracking or targeting cells presenting a particular antigen in vitro or in vivo, and may also be used as intermediates in the production of other multivalent specific binding molecule complexes having such uses.

Therapeutic agents that may be associated with the specific binding molecules of the present invention include immunomodulators and effectors, radioactive compounds, enzymes (e.g., perforins) or chemotherapeutic agents (e.g., cisplatin). To ensure therapeutic effect at the desired location, the agent may be located within a liposome or other nanoparticle structure linked to a specific binding molecule to provide for slow release of the compound. This will prevent destructive effects during in vivo transport and ensure that the agent will exert its maximum effect after the specific binding molecule binds to the relevant antigen presenting cell.

Examples of suitable therapeutic agents include, but are not limited to:

antibodies or fragments thereof, including anti-T cell or NK cell determinant antibodies (e.g., anti-CD 3, anti-CD 28, or anti-CD 16);

alternative protein scaffolds with antibody-like binding characteristics (e.g., DARPins);

immunostimulants, i.e. immune effectors that stimulate an immune response. Such as cytokines, e.g., IL-2 and IFN-gamma;

Chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory proteins, and the like;

Complement pathway activators or Fc receptors;

Checkpoint inhibitors, such as those targeting PD1 or PD-L1;

Small molecule cytotoxic agents, i.e., compounds having a molecular weight of less than 700 daltons that have the ability to kill mammalian cells. Such compounds may also contain toxic metals with cytotoxic effects. In addition, it is understood that these small molecule cytotoxic agents also include prodrugs, i.e., compounds that decay or transform under physiological conditions to release the cytotoxic agent. Examples of such drugs include cisplatin, maytansine derivatives, rapamycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sodium sofalcide photosensitizer II, temozolomide, topotecan, trimelimine, orestatin E vincristine, and doxorubicin;

peptide cytotoxins, i.e. proteins or fragments thereof that have the ability to kill mammalian cells. For example, ricin, diphtheria toxin, pseudomonas bacterial exotoxins A, dnase, and Rnase;

radionuclides, i.e., unstable isotopes of elements, which decay while emitting one or more alpha or beta particles or gamma rays. For example, iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225, and astatine 213; chelating agents may be used to facilitate the association of these radionuclides with TCRs or multimers thereof;

Superantigens and mutants thereof;

peptide-HLA complex, wherein the peptide is derived from a common human pathogen, such as Epstein Barr Virus (EBV)

Heterologous protein domain, allogeneic protein domain, viral/bacterial peptide.

Preferably, the soluble specific binding molecules of the invention are associated with immune effectors (typically by fusion to the N-or C-terminus of the alpha chain or beta chain, or both, in any suitable configuration). The N-terminus of the TCR may be linked to the C-terminus of the immune effector polypeptide.

Particularly preferred immune effectors are anti-CD 3 antibodies, or functional fragments or variants of said anti-CD 3 antibodies. Specific binding molecules of the invention comprising such antibodies are bispecific and may be referred to herein as "fusion molecules". As used herein, the term "antibody" encompasses such fragments and variants. Examples of anti-CD 3 antibodies include, but are not limited to OKT3, UCHT-1, BMA-031 and 12F6. Antibody fragments and variants/analogs suitable for use in the compositions and methods described herein include: minibodies, diabodies, fab fragments, F (ab') ₂ fragments, dsFv and scFv fragments. Other examples of what is encompassed within the term antibody include Nanobodies ^TM (these constructs are marketed by Ablynx (Belgium), comprising synthetic single immunoglobulin variable heavy domains derived from camelid (e.g., camel or llama) antibodies), domain Antibodies (domatis, belgium), comprising affinity matured single immunoglobulin variable heavy domains or immunoglobulin variable light domains, and alternative protein scaffolds exhibiting antibody-like binding properties, e.g., affibodies (Affibody, sweden), comprising engineered protein a scaffolds, or ANTICALINS (Pieris, germany), comprising engineered anti-calpain, or DARPins (Molecular Partners, switzerland), comprising engineered ankyrin repeats.

The anti-CD 3 antibody may be covalently linked to the C-or N-terminus of the TCR alpha or beta chain. The anti-CD 3 antibody may be covalently linked to the C-or N-terminus of the TCR β chain of the TCR by a linker sequence.

Preferably, the anti-CD 3 is a scFV fragment. Or the heavy and light chain variable domain fragments may be aligned in a diabody orientation. Particularly preferred anti-CD 3 sequences are provided in WO2020157210 and WO 2017163064.

Examples of preferred arrangements of fusion molecules include those described in WO2010133828, W02020157211, W02019012138 and W02019012141. The form described in WO2010133828 is particularly preferred.

Specific binding molecules of the invention may include:

a first polypeptide chain comprising an alpha chain variable domain and a first binding region of a variable domain of an antibody; and

A second polypeptide chain comprising a β chain variable domain and a second binding region of a variable domain of said antibody,

Wherein the individual polypeptide chains associate such that the specific binding molecule is capable of simultaneously binding to the SLYNTVATL-HLA-A2 complex ("SLYNTVATL" is disclosed as SEQ ID NO: 1) and the antigen of the antibody.

Also provided herein are bispecific polypeptide molecules selected from the group of molecules comprising a first polypeptide chain and a second polypeptide chain, wherein: a first polypeptide chain comprising a first binding region (VD 1) of the variable domain of an antibody which specifically binds to a cell surface antigen of a human immune effector cell, and

A first binding region (VR 1) of the variable domain of the TCR that specifically binds to an MHC associated peptide epitope, and

A first linker (LINK 1) connecting the domains;

A second polypeptide chain comprising a second binding region (VR 2) that specifically binds to a variable domain of a TCR of an MHC associated peptide epitope, and

A second binding region (VD 2) of the variable domain of an antibody that specifically binds to a cell surface antigen of a human immune effector cell, and

A second linker (LINK 2) connecting the domains;

Wherein the first binding region (VD 1) and the second binding region (VD 2) associate to form a first binding site (VD 1) (VD 2) for binding to a cell surface antigen of a human immune effector cell;

The first binding region (VR 1) associates with the second binding region (VR 2) to form a second binding site (VR 1) (VR 2) that binds to the MHC-related peptide epitope;

wherein the two polypeptide chains are fused to a human IgG hinge domain and/or a human IgG Fc domain or dimerizing portion thereof; and

Wherein the two polypeptide chains are linked by covalent and/or non-covalent bonds between the hinge domain and/or Fc domain; and

Wherein the bispecific polypeptide molecule is capable of binding to both a cell surface molecule and an MHC-related peptide epitope, and wherein the order of the binding regions in the two polypeptide chains is selected from VD1-VR1 and VR2-VD2 or VD1-VR2 and VR1-VD2 or VD2-VR1 and VR2-VD1, and wherein the domains are linked by LINK1 or LINK2, wherein the MHC-related peptide epitope is SLYNTVATL (SEQ ID NO: 1) complex and the MHC is HLA-A-02.

The attachment of the specific binding molecule and the anti-CD 3 antibody may be by covalent or non-covalent attachment. Covalent attachment may be direct or indirect via a linker sequence. Linker sequences are typically flexible in that they consist essentially of amino acids, such as glycine, alanine, and serine, which do not have bulky side chains that may limit flexibility. Or a more rigid joint may be desirable. The available length or optimal length of the linker sequence can be easily determined. The linker sequence is typically less than about 12 amino acids in length, for example less than 10 amino acids, or 2-10 amino acids. Examples of suitable linkers that may be used in the multidomain binding molecules of the invention include, but are not limited to ：GGGGS(SEQ ID NO:16)、GGGSG(SEQ ID NO:17)、GGSGG(SEQ ID NO:18)、GSGGG(SEQ ID NO:19)、GSGGGP(SEQ ID NO:20)、GGEPS(SEQ ID NO:21)、GGEGGGP(SEQ ID NO:22) and GGEGGGSEGGGS (SEQ ID NO: 23) (as described in WO 2010/133828) and GGGSGGGG (SEQ ID NO: 24). Other linkers may include sequences having one or more of the following sequence motifs: GGGS (SEQ ID NO: 25), GGGGS (SEQ ID NO: 26), TVLRT (SEQ ID NO: 27), TVSSAS (SEQ ID NO: 28) and TVLSSAS (SEQ ID NO: 29).

Preferred specific binding molecules of the invention have a beta chain comprising the amino acid sequence:

The surrogate specific binding molecules of the invention have a β chain comprising the amino acid sequence:

preferred alpha chains comprise the following amino acid sequences:

preferred specific binding molecules of the invention comprise the amino acid sequences of SEQ ID NOs 30 and 32.

Also included within the scope of the invention are functional variants (also referred to as phenotypically silent variants) comprising the specific binding molecules against CD 3.

In another aspect, the invention provides a nucleic acid encoding a TCR a chain and/or a TCR β chain of the invention. Nucleic acids encoding specific binding molecules of the invention, including such molecules fused to anti-CD 3 antibodies or fragments thereof, are also provided. In some embodiments, the nucleic acid is a cDNA. In some embodiments, the nucleic acid may be an mRNA, e.g., a bispecific molecule encoded by an mRNA (Stadler et al, nat Med.2017Jul;23 (7): 815-817). In some embodiments, the invention provides nucleic acids comprising sequences encoding TCR alpha chain variable domains of the specific binding molecules of the invention. In some embodiments, the invention provides nucleic acids comprising sequences encoding TCR β chain variable domains of the specific binding molecules of the invention. The nucleic acid may be non-naturally occurring and/or purified and/or engineered. The nucleic acid sequence may be codon optimized according to the expression system utilized. As known to those skilled in the art, expression systems may include bacterial cells, such as e.coli, or yeast cells, or mammalian cells, or insect cells, or they may be cell-free expression systems. In some embodiments, the molecule may be a bispecific antibody encoded by an mRNA.

In another aspect, the invention provides a vector comprising a nucleic acid of the invention. Preferably, the vector is a TCR expression vector. Suitable TCR expression vectors include, for example, gamma-retroviral vectors, or more preferably lentiviral vectors. For more details see Zhang 2012 and its references (Zhang et al, adv Drug Deliv Rev.2012Jun 1;64 (8): 756-762).

The invention also provides cells carrying the vectors of the invention, preferably TCR expression vectors. Suitable cells include mammalian cells, preferably immune cells, more preferably T cells. The vector may comprise a nucleic acid of the invention encoded in a single open reading frame, or two different open reading frames encoding the alpha and beta strands, respectively. Another aspect provides a cell carrying: a first expression vector comprising a nucleic acid encoding an alpha chain of a specific binding molecule of the invention, and a second expression vector comprising a nucleic acid encoding a beta chain of a specific binding molecule of the invention. Such cells are particularly useful in adoptive therapy. The cells of the invention may be isolated and/or recombinant and/or non-naturally occurring and/or engineered.

Since the specific binding molecules of the invention are useful in adoptive therapy, the invention includes non-naturally occurring and/or purified and/or engineered cells, particularly T cells presenting the specific binding molecules of the invention. The invention also provides an expanded population of T cells presenting the specific binding molecules of the invention. There are a number of methods suitable for transfecting T cells with a nucleic acid (e.g., DNA, cDNA or RNA) encoding a specific binding molecule of the invention (see, e.g., robbins et al, (2008) J Immunol. 180:6116-6131). T cells expressing the specific binding molecules of the invention will be suitable for use in adoptive therapy based cancer therapies. As known to those skilled in the art, there are many suitable methods by which adoptive therapy can be performed (see, e.g., rosenberg et al, (2008) NAT REV CANCER (4)).

As is well known in the art, in vivo production of proteins (including proteins comprising the specific binding molecules of the invention) may result in post-translational modifications. Glycosylation is a modification that involves covalent attachment of an oligosaccharide moiety to a particular amino acid in a polypeptide chain. For example, asparagine residues, or serine/threonine residues, are well known positions for oligosaccharide attachment. The glycosylation state of a particular protein depends on many factors, including protein sequence, protein conformation, and availability of certain enzymes. Furthermore, the glycosylation state (i.e., the total number of oligosaccharide types, covalent linkages, and attachments) can affect protein function. Thus, in the production of recombinant proteins, it is often desirable to control glycosylation. Controlled glycosylation has been used to improve antibody-based therapies (Jefferis et al, (2009) Nat Rev Drug Discov Mar;8 (3): 226-34). For the specific binding molecules of the invention, glycosylation can be controlled by use of specific cell lines, including, for example, but not limited to, mammalian cell lines, such as Chinese Hamster Ovary (CHO) cells or Human Embryonic Kidney (HEK) cells, or by chemical modification. Such modifications may be desirable because glycosylation may improve pharmacokinetics, reduce immunogenicity, and more closely mimic native human proteins (Sinclair and Elliott, (2005) Pharm Sci.Aug;94 (8): 1626-35). In some cases, mutations may be introduced to control and/or modify post-translational modifications.

For administration to a patient, the specific binding molecules of the invention (preferably associated with a detectable label or therapeutic agent such as anti-CD 3 or expressed on transfected T cells), the nucleic acids of the invention, the expression vectors of the invention, or the cells of the invention may be provided as part of a sterile pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. The pharmaceutical composition may be in any suitable form (depending on the method desired for administration to the patient). It may be provided in unit dosage form, will typically be provided in a sealed container, and may be provided as part of a kit. Such kits typically (but not necessarily) include instructions for use. It may comprise a plurality of said unit dosage forms.

The pharmaceutical composition may be adapted for administration by any suitable route, for example, parenteral (including subcutaneous, intramuscular, intrathecal or intravenous), enteral (including oral or rectal), inhalation or intranasal routes. Such compositions may be prepared by any method known in the pharmaceutical arts, for example by mixing the active ingredient with one or more carriers or excipients under sterile conditions.

The dosage of the substances according to the invention can vary within wide limits, depending on the disease or disorder to be treated, the age and condition of the individual to be treated, etc. Suitable dosages of the specific binding molecule-anti-CD 3 fusion molecule may range from 25ng/kg to 50. Mu.g/kg or from 1. Mu.g to 1 g. The physician will ultimately determine the appropriate dosage to be used. Examples of suitable dosing regimens are provided in WO 2017208018.

A single dose may be administered. Or multiple doses, e.g., two or more doses, may be administered; or three or more doses. When multiple doses are administered, the same dose may be administered each time, or a first and/or subsequent dose may be administered a reduced dose.

The specific binding molecules, pharmaceutical compositions, vectors, nucleic acids, and cells of the invention may be provided in substantially pure form, e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.

The invention also provides:

the specific binding molecules, nucleic acids, vectors, pharmaceutical compositions or cells of the invention are for use in medicine, preferably in a human subject, preferably in a method for treating HIV infection or AIDS in a human subject.

Use of a specific binding molecule, nucleic acid, vector, pharmaceutical composition or cell of the invention in the manufacture of a medicament for treating HIV infection or aids in a human subject.

A method of treating HIV infection or aids comprising administering to a subject in need thereof a therapeutically effective amount of a specific binding molecule, nucleic acid, vector, pharmaceutical composition or cell of the invention.

An injectable formulation for administration to a human subject comprising a specific binding molecule, nucleic acid, vector, pharmaceutical composition or cell of the invention.

The specific binding molecules, nucleic acids, vectors, pharmaceutical compositions or cells of the invention may be administered by injection or infusion. Particular preference is given to administration by intravenous infusion or subcutaneous injection. The human subject may be of subtype HLA-A x 02.

The method of treatment may further comprise the separate, combined or sequential administration of one or more additional antiviral agents and/or one or more additional immunotherapeutic agents and/or one or more soluble bispecific binding proteins. For example, a method of treatment may comprise administering two or more bispecific binding proteins, including a specific binding molecule of the invention and an additional specific binding molecule that recognizes a surrogate HIV protein.

Patients receiving treatment may be receiving antiretroviral therapy (ART). Or the patient receiving treatment may have stopped using ART, for example, such patient may be receiving an Analytical Therapy Interruption (ATI). Or the patient receiving treatment may not have received ART.

The terms "treatment", "treatment" and "treatment" are intended to include slowing, stopping or reversing the progression of HIV and/or AIDS. These terms also include alleviating, ameliorating, reducing, eliminating, or alleviating one or more symptoms of a disease or disorder, even if the disease or disorder is not actually eliminated, even if the progression of the disease or disorder itself is not slowed, stopped, or reversed.

"Therapeutically effective amount" refers to the amount of a compound, or pharmaceutically acceptable salt thereof, that will elicit the biological or medical response or the desired therapeutic effect in a subject upon administration to the subject.

The therapeutically effective amount can be readily determined by one skilled in the art using known techniques and by observing results obtained in similar circumstances. In determining an effective amount of a subject, the attending physician may consider a number of factors, including, but not limited to: body type, age, and general health; the particular disease or condition involved; the extent or severity of the disease or condition; a response of the individual subject; the particular compound administered; the mode of administration; bioavailability characteristics of the administered formulation; a selected dosage regimen; simultaneously using medicines; and other related conditions.

The preferred features of each aspect of the invention are applicable to each of the other aspects as well as mutatis mutandis. The prior art documents mentioned herein are incorporated by reference to the maximum extent allowed by law.

The invention is further illustrated by the following non-limiting examples. Reference is made to the accompanying drawings.

Examples

Example 1 identification of Single TCR alpha chain mutations that increase yield without adversely affecting target binding affinity

A bispecific molecule comprising a high affinity HIV TCR sequence fused to an anti-CD 3 scFv was produced according to the methods described previously (Yang et al, mol Ther.2016;24 (11): 1913-1925; WO 2017163064). The TCR end of this molecule recognizes HLA-A.times.02 restriction 9-mer peptide SLYNTVATL (SEQ ID NO: 1) derived from the HIV Gag protein (Gag _78-85). The sequence of the TCR domain is shown in FIG. 1a (SEQ ID NOS: 33 and 34) and the sequence of the complete bispecific molecule is shown in FIG. 1b (SEQ ID NOS: 35 and 36). During preclinical studies, the yield of this molecule was found to be low (-1 mg/L) when produced in E.coli. Production yields in this range are considered unsuitable for pharmaceutical preparation to support clinical development of the molecule. Attempts to increase yield by optimizing refolding and purification conditions using standard methods have been unsuccessful. Unexpectedly, it was found that a single mutation in the variable domain of the TCR a chain increases yield without decreasing binding affinity to the cognate pMHC target.

Method of

Single point mutagenesis was performed to introduce a F to K mutation at position 50 of the TCR alpha chain variable domain, the sequence of which is shown in FIG. 2 (NB-in this example, the residue numbering is based on the inclusion of an N-terminal methionine). Bispecific proteins containing this mutation were produced in E.coli. The yield and binding parameters of the mutants were compared to the corresponding non-mutated versions.

The preparation of bispecific proteins is described above. Briefly, the alpha and beta chains were expressed as inclusion bodies in E.coli strain BL21-DE3 (pLysS), respectively, by induction with 0.5mM IPTG in mid-log phase. Inclusion bodies were isolated by sonication and then subjected to successive washing and centrifugation steps using 0.5% Triton X-100. Soluble proteins were refolded by rapid dilution of a mixture of solubilized alpha and beta chain inclusion bodies in 5M urea, 0.4M L-arginine, 100mM Tris pH 8.1, 3.7mM cystamine, 6.6mM b-mercaptoethylamine. The refolded mixture was dialyzed against 10 volumes of deionized water for 24 hours and then against 10 volumes of 10mM Tris pH 8.1. Refolded proteins were filtered and purified by three sequential chromatography steps-anion exchange (AIEX), cation exchange (CIEX) and Size Exclusion (SEC) pre-equilibrated in Phosphate Buffered Saline (PBS). Fractions containing the main peak were pooled and further analyzed. The final purified molecules were analyzed by SDS-PAGE under reducing and non-reducing conditions. The yields and recovery of all purification steps (AIEX, CIEX and SEC) were determined.

As described previously, use ofThe system performs a Surface Plasmon Resonance (SPR) analysis (see, e.g., yang et al, mol Ther.2016;24 (11): 1913-1925; WO 2017163064) on the purified bispecific molecule to determine binding of the TCR end of the molecule to its target peptide HLA complex (SLYNTVATL (SEQ ID NO: 1) HLA-A.02). Briefly, biotinylated pHLA was immobilized on a streptavidin-coupled CM5 sensor chip. Flow cell one was loaded with free biotin alone as a control surface. Suppose Langmuir binding calculates K _D values and data is analyzed using a 1:1 binding model (Biacore Insight Evaluation v2.0.15.12933 for single cycle kinetic analysis).

Results

Table a shows the overall yield and recovery for all purification steps. FIG. 3 shows the yield per volume of culture. For F50K, the overall yield and% recovery for all purification steps was significantly improved over the non-mutated (WT) version; furthermore, for F50K, the yield per volume of culture was increased by a factor of 4.5 relative to the wild type. Table B shows the target binding parameters for each bispecific protein. These data indicate that F50K retains similar binding properties to non-mutant WT proteins, including K _D and T1/2 (K _D =low pM and T1/2= >24 hours).

As a comparison, three additional single point mutations (L47P, M, 49, K, A E) were made in the same region as F50 and tested using the same method. As shown in Table A and FIG. 3, the mutations L49P and A53E did not result in a significant increase in yield compared to F50K, whereas M49K produced slightly higher (4.9-fold) than F50K. Interestingly, all three substitution mutations, including M49K, showed a significant decrease in binding properties relative to the WT target. Figure 4 shows a side-by-side comparison of binding kinetics for M49K and F50K.

TABLE A Total yield and recovery of bispecific proteins after each chromatography step

Table B-binding parameters for each bispecific protein as determined by SPR

These data demonstrate the challenge of identifying bispecific proteins with improved developability. A single mutation in the variable domain of the TCR alpha chain (F50K) has been demonstrated to both increase yield and maintain high affinity binding to the target.

Example 2 identification of additional TCR alpha chain mutations that increase protein stability without adversely affecting target binding affinity

Further evaluation of the F50K bispecific protein indicated that reduced stability may affect the developability of the molecule. Unexpectedly, a single mutation in the variable region of the TCR alpha chain was found to increase the stability of the protein.

Method of

Single point mutagenesis was performed to introduce S to A mutations at position 96 of the TCR alpha chain variable domain as shown in FIG. 5 (NB-in this example, residue numbering is based on the inclusion of an N-terminal methionine). Bispecific proteins containing this mutation were produced in E.coli and the binding parameters were analyzed as described previously.

Protein stability after three days exposure to high pH (Tris pH 9) was assessed using analytical anion exchange chromatography (AIEX-UPLC). Proteins were separated on anion exchange UPLC columns, eluted in order of increasing net surface negative charge, and detected by FLD detection (excitation 295nm, emission 348 nm). The charge distribution of the test sample was monitored by integrating the area under the curve of all peaks. The relative decrease in the main peak and the corresponding increase in the acidic species are used as a measure of stability. Deamidation was assessed using a non-reducing peptide profile. Briefly, at +37℃, the test sample and reference protein were denatured and digested with a combination of trypsin and lysC. The resulting peptides were separated on a C18 HPLC column and detected by uv absorbance at 214 nm. Using PMI Byonic software, the library of potential peptides was searched for the resulting fragmentation spectrum for each peptide based on protein sequence, mass accuracy and potential modification.

Results

Table C shows the target binding parameters. These data indicate that S96A produces similar target binding parameters compared to F50K alone. As a comparison, three alternative single point mutations were introduced in the region of the same protein as S96A. Wherein, compared to F50K alone, only T94L produced similar target binding parameters, whereas N95Q or S96V did not. Table C-binding parameters for each bispecific protein as determined by SPR

Table D summarizes the results of the AIEX-UPLC analysis. These data indicate that S96A is more stable than F50K or T94L alone. Further analysis of S96A and T94L by peptide mapping showed that the improvement in stability may be a result of reduced deamidation at the N95 position.

Table D-AE-UPLC analysis

TABLE E-peptide map

Example 3-improved bispecific molecule recognizes common viral escape variants and exhibits potent and specific killing of HIV-infected cells

Bispecific proteins that bind f50k+s965a were also analyzed to assess further properties, including recognition of common viral escape variants and efficacy against antigen positive cell lines. In this example, the TCR is fused to an alternative anti-CD 3 domain, which is further described in WO 2020157210. The complete sequence of the bispecific molecule is shown in figure 6.

Method of

SPR analysis was performed on Biacore equipment as described previously. Briefly, variants of the HIV peptides listed herein were complexed in soluble form with HLAA 02:01; these biotinylated complexes were then immobilized on streptavidin-preloaded Biacore CM5 chips. Soluble bispecific protein was injected onto the immobilized pHLA at 5 increasing concentrations. Dissociation was measured for 2 hours after the fifth injection. Binding and dissociation of molecules to each HLA complex was measured using Biacore equipment. The relative half-life of each interaction was calibrated according to the index peptide in HLAA 02:01.

Cytokine enzyme-linked immunosorbent spot (ELISPOT) assays detect interferon gamma (IFN-gamma) or granzyme B (GrB) secreted after T cell activation to determine efficacy. HLAA 02:01 lymphoma derived cancer cell line T2 (pulsed with HIV Gag _{77_85} peptide) and peptide pulsed HLAA 02:01/β2m transduced T cell line (C8166 A2B 2M) were used as target cells for IFN- γ and GrB assays, respectively. PBMCs obtained from uninfected HIV donors were used as effectors. Cytokine release (IFN- γ) was used to measure T cell activation and GrB release was used as an alternative indicator of T cell mediated killing of target cells. Target cells were incubated with PBMCs from different uninfected HIV donors (CTL 013, CTL014, CTL018, SC009, SC 001B) and increasing concentrations of IMCM113V, overnight (IFN- γ specific, top) or 4048 hours (GrB specific, bottom).

Results

Table F shows that bispecific proteins bind to each of the variant peptides tested with affinities (K _D) in the low nanomolar to picomolar range and half-lives (t 1/2) of several hours. Although there was a decrease in affinity for variants, particularly those comprising Y3F and T8V substitutions, the weakest interaction between IMCM113V and the peptide HLA complex (Y3F T V) was still considered very strong relative to the wild-type TCR. These data indicate that bispecific proteins can strongly recognize common escape variants.

Table F-binding parameters of bispecific proteins and peptide variants

FIG. 7 shows that bispecific protein redirecting effector T cells release IFN-. Gamma.and GrB in a dose dependent manner when incubated with HLAA 02:01 positive peptide (HIV Gag _{77_85}) pulsed T2 and C8166A 2B2M target cells. The EC ₅₀ values obtained from the responding donors are shown graphically, ranging from 1.0 to 12.3pM for IFN-gamma release and from 0.5 to 81.1pM for GrB release. These data indicate that in the presence of HLAA 02:01 positive Gag _{77_85} positive cells, bispecific proteins can specifically redirect T cell activity to activate T cells, resulting in dose dependent cytokine release, and efficacy in the low picomolar range.

Claims (24)

1. A specific binding molecule having the property of binding to SLYNTVATL (SEQ ID NO: 1) in complex with HLA-A*02, and comprising a TCR α chain variable domain and a TCR β chain variable domain, Wherein the α chain variable domain comprises an amino acid sequence selected from the group consisting of: a) b) c) optionally having an N-terminal methionine (SEQ ID NOs: 41-43), and The beta chain variable domain comprises the following amino acid sequence: Optionally has an N-terminal methionine (SEQ ID NO: 44). 2. The specific binding molecule according to claim 1, wherein the α chain variable domain comprises the amino acid sequence: 3. The specific binding molecule according to claim 1, wherein the α chain variable domain comprises the amino acid sequence: AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMFL YADPDKEDGRFTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 3). 4. The specific binding molecule according to claim 1, wherein the α chain variable domain comprises the amino acid sequence: AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYS GKSPELIMKL YADPDKEDGRFTAQLNKASQ YVSLLIRDSQ PSDSATYLCA VRTNAGYALN FGKGTSLLVT P (SEQ ID NO: 4). 5. A specific binding molecule according to any one of the preceding claims, wherein the α chain variable domain amino acid sequence comprises one or more of the following mutations: A2Q V73I Q81K P82L. 6. The specific binding molecule according to any one of the preceding claims, which is an alpha-beta heterodimer having an alpha chain TRAC constant domain and a beta chain TRBC1 or TRBC2 constant domain. 7. The specific binding molecule according to claim 6, wherein the amino acid sequences of the α and β chain constant domains are modified by truncation or substitution to delete the native disulfide bond between Cys4 of TRAC exon 2 and Cys2 of TRBC1 or TRBC2 exon 2. 8. A specific binding molecule according to claim 6 or 7, wherein the α and β chain constant domain amino acid sequences are modified by replacing Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2 with cysteine residues, which form a disulfide bond between the TCR α and β constant domains. 9. A specific binding molecule according to any of the preceding claims, which is a single-chain form of the Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, Vα-Cα-L-Vβ-Cβ or Vβ-Cβ-L-Vα-Cα type, wherein Vα and Vβ are TCRα and β variable domains, respectively, Cα and Cβ are TCRα and β constant domains, respectively, and L is a linker sequence. 10. The specific binding molecule according to any one of the preceding claims, associated with a detectable label, a therapeutic agent or a PK modifying moiety. The specific binding molecule according to claim 10 , which is associated with an anti-CD3 antibody covalently linked to the C-terminus or N-terminus of the TCR α or β chain. 12 . The specific binding molecule according to claim 11 , wherein the anti-CD3 antibody is covalently linked to the C-terminus or N-terminus of the TCRβ chain via a linker sequence. 13. The specific binding molecule of claim 12, wherein the linker sequence is selected from the group consisting of GGGGS (SEQ ID NO: 16), GGGSG (SEQ ID NO: 17), GGSGG (SEQ ID NO: 18), GSGGG (SEQ ID NO: 19), GSGGGP (SEQ ID NO: 20), GGEPS (SEQ ID NO: 21), GGEGGGP (SEQ ID NO: 22), and GGEGGGSEGGGS (SEQ ID NO: 23). 14. The specific binding molecule according to claim 12 or 13, wherein the beta chain comprises the amino acid sequence: AIQMTQSPS SLSASVGDRV TITCRASQDI RNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGT DYTLTISSLQPEDFATYYCQ QGNTLPWTFG QGTKVEIKGG GGSGGGGSGGGGSGGGGSGGGSEVQLVESG GGLVQPGGSL RLSCAASGYSFTGYAMNWVR QAPGKGLEWV ALINPYKGVSTYN QKFKDRFTFSVDKSKNT AYLQMNSLRA EDTAVYYCAR SGYYGDSDWYFDVWGQGTLV TVSSGGGGSDAGVTQSPTHL IKTRGQQVTLRCSPKSGHDT VSWYQQALGQ GPQFIFQAVR GVERQRGNFPDRFSGHQFPNYSSELNVNAL LLGDSALYLC ASSDTVSYEQYFGPGTRLTV TEDLKNVFPP EVAVFEPSEAEISHTQKATLVCLATGFYPD HVELSWWVNG KEVHSGVCTD PQPLKEQPALNDSRYALSSR LRVSATFWQDPRNHFRCQVQ FYGLSENDEWTQDRAKPVTQ IVSAEAWGRA D (SEQ ID NO: 30). 15. The specific binding molecule according to claim 12 or 13, wherein the beta chain comprises the amino acid sequence: AIQMTQSPS SLSASVGDRV TITCRASQDI RNYLNWYQQKPGKAPKLLIY YTSRLESGVPSRFSGSGSGT DYTLTISSLQPEDFATYYCQ QGNTLPWTFG QGTKVEIKGG GGSGGGGSGGGGSGGGGSGGGSEVQLVESG GGLVQPGGSL RLSCAASGYSFTGYTMNWVR QAPGKGLEWV ALINPYKGVSTYN QKFKDRFTISVDKSKNT AYLQMNSLRA EDTAVYYCAR SGYYGDSDWYFDVWGQGTLV TVSSGGGGSDAGVTQSPTHL IKTRGQQVTLRCSPKSGHDT VSWYQQALGQ GPQFIFQAVR GVERQRGNFPDRFSGHQFPNYSSELNVNAL LLGDSALYLC ASSDTVSYEQYFGPGTRLTV TEDLKNVFPP EVAVFEPSEAEISHTQKATLVCLATGFYPD HVELSWWVNG KEVHSGVCTD PQPLKEQPALNDSRYALSSR LRVSATFWQDPRNHFRCQVQ FYGLSENDEWTQDRAKPVTQ IVSAEAWGRA D (SEQ ID NO: 31). 16. The specific binding molecule according to claim 14 or 15, wherein the α chain comprises the amino acid sequence: AKEVEQNSGP LSVPEGAIAS LNCTYSSWEG QSFFWYRQYSGKSPELIMKL YADPDKEDGRFTAQLNKASQ YVSLLIRDSQPSDSATYLCA VRTNAGYALN FGKGTSLLVT PHIQKPDPAVYQLRDSKSSDKSVCLFTDFD SQTNVSQSKD SDVYITDKCVLDMRSMDFKS NSAVAWSNKSDFACANAFNN SIIPEDT(SEQID NO :32). 17. A nucleic acid molecule encoding a TCR alpha chain and/or a TCR beta chain as defined in any preceding claim. An expression vector comprising the nucleic acid according to claim 17 . 19. A cell carrying (a) a TCR expression vector comprising the nucleic acid according to claim 17 in a single open reading frame, or two different open reading frames encoding the α chain and the β chain, respectively; or (b) a first expression vector comprising a nucleic acid encoding the α chain of the TCR according to any one of claims 1 to 16, and a second expression vector comprising a nucleic acid encoding the β chain of the TCR according to any one of claims 1 to 16. 20. An isolated or non-naturally occurring cell, in particular a T cell, presenting a TCR according to any one of claims 1 to 10. 21. A pharmaceutical composition comprising a specific binding molecule according to any one of claims 1 to 16, a nucleic acid according to claim 17, a vector according to claim 18 and/or a cell according to claim 19 or 20, and one or more pharmaceutically acceptable carriers or excipients. 22. A specific binding molecule according to any one of claims 1 to 16, a nucleic acid according to claim 17, a vector according to claim 18 and/or a cell according to claim 19 or claim 20 for use in medicine in a human subject. 23. A specific binding molecule according to any one of claims 1 to 16, a nucleic acid according to claim 17, a vector according to claim 18 and/or a cell according to claim 19 or claim 20 for use in a method for treating HIV infection or AIDS in a human subject. 24. A method of treating HIV infection or AIDS in a human subject comprising administering a therapeutically effective amount of a specific binding molecule according to any one of claims 1 to 16.