HER2 variant CAR

Abstract

The present invention provides a binding molecule comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) which together form an antigen-binding unit, comprising the amino acid sequence set forth in SEQ ID NO: 17, which specifically binds to p95HER2, wherein the VL comprises three complementarity determining regions (CDRs), i.e., CDR1, CDR2 and CDR3, comprising the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively, and the VH comprises three CDRs, i.e., CDR1, CDR2 and CDR3, comprising the amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively.

Description

[Technical field]

[0001] The present invention relates to the field of cancer therapy and diagnosis. In particular, the present invention relates to a novel targeting unit and a chimeric antigen receptor (CAR) comprising the same, a nucleic acid encoding the targeting unit, a nucleic acid encoding the CAR, an immune cell expressing the CAR, and their usefulness for the treatment of cancer. [Background technology]

[0002] Many HER2+ breast cancers express isoforms of HER2 that have truncated carboxy-terminal fragments (CTFs), collectively referred to as p95HER2.

[0003] Although several antigen binding units that bind to p95HER2 are known, not all of them are suitable for implementation into CARs, as shown by the failures described in research disclosure RD667070 published on October 17, 2019.

[0004] To achieve therapeutic CAR-T cells (CAR-T), the cells need to express sufficient amounts of CAR on the cell membrane and the antigen-binding unit needs to mediate sufficient affinity and specificity for the target antigen. It can be expected that only a small proportion of CAR-T cells with in vitro activity will be successful in migrating to tumor metastases and / or infiltrating the hostile tumor microenvironment of solid tumors in vivo. Furthermore, CAR-T cells likely need to sustain their activity over time to exert a therapeutic effect in vivo. Therefore, obtaining novel CARs that can provide a therapeutic effect against solid tumors in vivo when the CAR is expressed on the cell membrane of immune cells is not trivial and is highly desirable. Summary of the Invention [Problem to be solved by the invention]

[0005] A binding molecule comprising a novel antigen-binding unit is provided herein. The antigen-binding unit and the binding molecule comprising it can specifically bind to cells expressing the hyperactive 611-CTF isoform of p95HER2 under physiological conditions. Since p95HER2 is a truncated transmembrane receptor, it is not trivial to obtain such an antigen-binding unit. The antigen-binding unit of the present invention shows little or no binding to full-length HER2 under physiological conditions and shows little or no cross-reactivity with healthy tissues. The antibody comprising the novel antigen-binding unit showed an affinity (KD) of approximately 2nM for p95HER2.

[0006] As demonstrated herein, the antigen-binding unit is suitable for implementation into a CAR and in that format retains sufficient affinity and specificity for p95HER2, the CAR is fully expressed in T cells, its functionality is confirmed by in vitro experiments, and its therapeutic effect is demonstrated in an in vivo model. [Means for solving the problem]

[0007] In a first aspect, a binding molecule is provided which specifically binds to p95HER2 comprising a VL and a VH which together form an antigen-binding unit comprising the amino acid sequence set forth in SEQ ID NO: 17, wherein the VL comprises three complementarity determining regions (CDRs), i.e. CDR1, CDR2 and CDR3, comprising the amino acid sequences SEQ ID NOs: 1, 2 and 3, respectively, and the VH comprises three CDRs, i.e. CDR1, CDR2 and CDR3, comprising the amino acid sequences SEQ ID NOs: 4, 5 and 6, respectively.

[0008] The binding molecule may comprise a VH comprising the amino acid sequence set forth in SEQ ID NO: 7, or a sequence having at least 90% identity thereto, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 8, or a sequence having at least 90% identity thereto. The antigen-binding unit may be an scFv.

[0009] In a second aspect, there is provided a chimeric antigen receptor (CAR) comprising an antigen-binding unit according to the first aspect. The CAR may comprise a human CD8α hinge.

[0010] A CAR may comprise, from N-terminus to C-terminus, a human CD8α hinge, a human CD8α transmembrane domain, a human 4-1BB costimulatory domain, and a human CD3ζ signaling domain.

[0011] In a third aspect, there is provided a nucleic acid encoding a binding molecule according to the first aspect or a CAR according to the second aspect.

[0012] In a fourth aspect, there is provided a vector comprising a nucleic acid of the third aspect.

[0013] In a fifth aspect, there is provided a cytotoxic immune cell expressing a CAR according to the second aspect in its cell membrane.

[0014] In a sixth aspect, there is provided a pharmaceutical composition comprising a binding molecule according to the first aspect, a nucleic acid according to the third aspect, a vector according to the fourth aspect, or a cytotoxic immune cell according to the fifth aspect.

[0015] In a seventh aspect, there is provided a method of treating cancer in a human patient comprising administering a cytotoxic immune cell of the fifth aspect or a pharmaceutical composition of the sixth aspect.

[0016] In an eighth aspect, there is provided a method of treating cancer in a human patient, the method comprising the steps of: (a) obtaining a sample from the patient comprising cancer cells; (b) analysing whether the cancer cells express p95HER2 by contacting the cells ex vivo with a binding molecule according to the first aspect, the binding molecule further comprising a moiety suitable for detection; and (c) administering an approved chemotherapy to the patient if the cancer cells are p95HER2 positive.

[0017] In a ninth aspect, there is provided a method for diagnosing cancer comprising the steps of: (a) obtaining a sample comprising cells from a human patient; (b) analysing whether the cells express p95HER2 by contacting the cells ex vivo with a binding molecule according to the first aspect comprising a moiety suitable for detection; and (c) diagnosing the patient with cancer if the cells express p95HER2.

[0018] In a tenth aspect, there is provided a binding molecule according to the first aspect, a CAR according to the second aspect, a cytotoxic immune cell according to the fifth aspect, or a pharmaceutical composition according to the sixth aspect for use in therapy.

[0019] In an eleventh aspect, there is provided a binding molecule according to the first aspect, a CAR according to the second aspect, a cytotoxic immune cell according to the fifth aspect, or a pharmaceutical composition according to the sixth aspect for use in the treatment of a cancer expressing p95HER2.

[0020] In a twelfth aspect, there is provided a method of diagnosing cancer in a subject, the method comprising the steps of: (a) contacting a sample of cells from the subject with a binding molecule according to any one of claims 1 to 4, the binding molecule further comprising a detection moiety; (b) determining whether the cells express p95HER2; and (c) diagnosing the patient with cancer if the cells express p95HER2. Thus, the method of the twelfth aspect is an ex vivo method performed on a sample. [Brief description of the drawings]

[0021] [Figure 1]Figure 1 shows the reactivity and specificity of the antibody against p95HER2. The reactivity of the antibody of interest was tested using flow cytometry against a panel of 15 HER2+ / - cell lines. The antibody bound only to the p95HER2-T47D cell line and was not reactive to cell lines expressing full-length HER2 (HER2+ SK-BR-3, MDA-MB-468 and A549) and the HER2- breast cancer cell lines T47D, MCF-7 and MDA-MB-231. The antibody was also not reactive to any of the other malignant cell lines tested. [Diagram 2] Figure 2 shows the antibody binding affinity. To determine the binding affinity of the antibodies, p95HER2 peptide was used as analyte in serial dilutions from 0.6 to 2500 nM. A control antibody against the HER2 cytoplasmic domain (as a reference) and the antibody of interest were covalently immobilized on two different flow cell surfaces on a sensor chip. The association (ka) rate increased with increasing p95HER2 peptide concentration. A bimolecular interaction model 1:1 showed a low equilibrium dissociation constant (KD=2 nM) with a high maximum binding response (Rmax) of 137 RU for the antibody of interest. Two independent experiments were performed. [Figure 3-1] Figure 3 is a diagram of epitope mapping. (a) Map of consecutive overlapping synthetic peptides (triplets) based on the p95HER2 extracellular domain. The consecutive peptides share 11 aa. (b) Based on signal intensity, the antibody of interest was only reactive to peptide 1, the only peptide containing MPIW (SEQ ID NO: 33) (c) that is highlighted in the 3D structure of full-length HER2. [Figure 3-2] (d) Maps of C-terminally extended p95HER2 peptides that are truncated from the N-terminus (peptides 1–20, each peptide 1 aa shorter than the previous peptide) or have two amino acids replaced with alanine (peptides 21–39). [Figure 3-3](e) Signal intensity from the truncated peptides indicated that the W-axis PIW was important for antibody binding, while substitutions revealed that KFPDEE (SEQ ID NO: 34) was also required for antibody binding. Sequential overlap blots contain triplicates from each peptide, and truncation and substitution blots contain duplicates from each peptide. [Figure 4] Figure 4. (A) visualizes the percentage of T cells transduced with the p95HER2 CAR construct compared to non-transduced T cells (NT). (B) demonstrates the in vitro cytotoxicity of p95HER2-CAR-T. p95HER2-CAR-T cells, control CD19-CAR-T cells, and non-transduced T cells were co-cultured with T47D-p95HER2 and T47D (p95HER2 negative) cells. p95HER2-CAR-T induced apoptosis in over 90% of T47D-p95HER2 target cells overnight, but did not have any effect on T47D cells. Similarly, no cytotoxicity was observed for CD19-CAR-T or non-transduced T cells co-cultured with T47D-p95HER2 or T47D cells. CD19-CAR-T contains a CD19-specific scFv cloned into the same CAR backbone as the p95HER2 CAR, with identical hinge, transmembrane, and intracellular domains. [Diagram 5] Figure 5 visualizes cytokine production of p95HER2-CAR-T. Both CD4+ and CD8+ p95HER2-CAR-T compartments expressed significantly higher levels of TNFα and IFNγ when co-cultured with p95HER2-expressing T47D cells compared to p95HER2-negative T47D cells. Non-transduced T cells did not express any cytokines when co-cultured with either p95HER2-positive or p95HER2-negative T47D. [Figure 6]Figure 6 visualizes the antitumor effect of p95HER2-CAR-T in vivo. Tumor control of p95HER2-CAR-T and CD19-CAR-T on tumor growth in an orthopedic p95HER2 breast cancer-bearing NSG mouse model was evaluated by in vivo bioluminescence imaging. CAR-T was intravenously injected twice (2 and 5 weeks after tumor implantation, indicated by arrows) with 5 million cells at each time point. p95HER-CAR-T showed significant tumor control only 2 weeks after the first injection and completely eradicated p95HER+ tumors 5 weeks after the first injection. CD19-CAR-T did not show any effect on p95HER+ tumor progression. There was no difference in tumor growth between the CD19-CAR-T and tumor-only groups. Data are reported as ± SEM of a representative experiment out of 3 independent replicates (n=10). [Figure 7] Figure 7 visualizes the number of CAR-T in 1 μl of circulating blood. The number of p95HER2-CAR-T increased over time through activation by interaction with target cells expressing the p95HER2 antigen. p95HER2-CAR-T persisted in vivo for more than 10 weeks after intravenous injection. The same trend was not observed for CD19-CAR-T through in vivo interaction with target cells expressing the p95HER2 antigen. Female mice, on average, have a circulating blood volume of 2.5 to 3.75 ml. [Figure 8] Figure 8. Some breast cancer cells express isoforms of HER2 (sequence shown - SEQ ID NO: 71) that are generated through two different mechanisms. Proteolytic cleavage of HER2 by metalloproteinases was the first mechanism discovered. The second mechanism involves alternative translation initiation from internal methionine codons located at positions 611, 648, 676 or 687. Several isoforms with different states of activity have been identified and are collectively referred to as p95HER2. The most potent and hyperactive p95HER2 isoform is called 611-HER2-CTF (carboxy terminal fragment). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Binding molecules comprising the novel antigen-binding units herein generally comprise or consist of one or more proteins (i.e., polypeptide chains) and can have any suitable format, including antibodies, scFvs, Fabs, immunotoxins, immunoconjugates, bispecific antibodies, CARs, etc. Thus, in one embodiment, the binding molecules provided herein are antibodies or fragments thereof (i.e., antigen-binding fragments). Examples of antigen-binding fragments of antibodies include Fab, Fab', and F(ab)'2 portions. In other embodiments, the binding molecule is an scFv. In yet other embodiments, the binding molecule is a CAR.

[0023] The binding molecules provided herein specifically bind to p95HER2, which comprises the amino acid sequence PIWKFPDEE set forth in SEQ ID NO: 17. As described in further detail below, SEQ ID NO: 17 is the epitope recognized by the binding molecules provided herein.

[0024] Such binding molecules, particularly soluble binding molecules such as antibodies, antigen-binding fragments of antibodies, and scFvs, can be used in their "naked" form (i.e., not conjugated to a second agent) to target cancer cells. Alternatively, such binding molecules can be conjugated to a toxic payload, such as a cytotoxin (such as saporin or gelonin), or a radioisotope, such as 177 Lu, 224 Ra or 225 It may carry (e.g., be conjugated to) a moiety including Ac, etc. A binding molecule conjugated to a toxic payload may be referred to as an immunotoxin.

[0025] Furthermore, the novel antigen-binding units can be used as diagnostic agents, for example in the form of naked antibodies or binding molecules that include a detectable label, such as a fluorescent or radioactive moiety. A detectable label can be referred to as a detection moiety or a moiety suitable for detection.

[0026] For an antibody comprising a heavy chain variable region (VH) (SEQ ID NO: 7) and a light chain variable region (VL) (SEQ ID NO: 8), a low equilibrium dissociation constant (KD=2 nM) was measured with a high maximum binding response (Rmax) of 137 RU. The target epitope of the antigen-binding unit herein is believed to be the sequence PIWKFPDEE (SEQ ID NO: 17). The epitope is located in the p95HER2 isoform, designated 611-HER2-CTF (SEQ ID NO: 20). (SEQ ID NO:20)

[0027] Thus, an antigen-binding unit is provided that can specifically bind to the sequence PIWKFPDEE (SEQ ID NO: 17) under physiological conditions. In one embodiment, a binding molecule, e.g., an antibody, comprising an antigen-binding unit provided herein has a KD of at least 2 nM.

[0028] As used herein, an "antigen binding unit" is a moiety that comprises or consists of one or more proteins or portions thereof that can bind to an extracellular target epitope under physiological conditions. The antigen binding unit of the present invention may be capable of binding to an extracellular target epitope under physiological conditions in a tumor environment. The antigen binding unit of the present invention can specifically bind to p95HER2 expressed on cancer cells under physiological conditions. That is, the antigen binding unit of the present invention exhibits little or no binding to full-length HER2 under physiological conditions. Furthermore, the antigen binding unit of the present invention exhibits little or no cross-reactivity with healthy tissues.

[0029] In particular, the antigen-binding units herein can bind to epitopes that are masked in full-length HER2 but exposed in 611-CTF, making them highly specific for the hyperactive p95HER2 isoform, which is the only known isoform of p95HER that broadly induces the expression of genes involved in the metastasis and development of malignant tumors.

[0030] Thus, binding molecules comprising the antigen binding units provided herein specifically bind to p95HER2 comprising the amino acid sequence set forth in SEQ ID NO: 17 and can therefore bind (or target) cancer cells expressing a p95HER2 isoform (such as p95HER2-611-CTF) that comprises the epitope of SEQ ID NO: 17. In particular, CARs provided herein that comprise such antigen binding units can target cytotoxic cells expressing the CAR to such cancer cells in order to destroy those cancer cells.

[0031] The antigen-binding unit provided herein comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH). Such variable domains are well known to those skilled in the art. An antibody antigen-binding unit comprising VL and VH is called Fv. An antigen-binding unit as defined herein may comprise a single polypeptide chain comprising both VL and VH sequences (e.g., in the case of scFv), or VL and VH may be provided on separate polypeptide chains (in the case of Fv).

[0032] Each VL and VH herein comprises three complementarity determining regions (CDRs) flanked by framework sequences. The framework sequences may be human, humanized, or murine. The six CDRs are represented by the following sequences: VL CDR1 (SEQ ID NO: 1): KSSQSLLSSGNQKNNLA VL CDR2 (SEQ ID NO: 2): YASTRQS VL CDR3 (SEQ ID NO: 3): LQHYSSPYT VH CDR1 (SEQ ID NO: 4): DYFMN VH CDR2 (SEQ ID NO: 5): QIRNKNYNYATYFAESLEG VH CDR3 (SEQ ID NO: 6): LRYDY It comprises or consists of.

[0033] The CDR sequences specified above were determined using the Kabat system.

[0034] The Framework1 sequence is N-terminal to CDR1, the Framework2 sequence is located between CDR1 and CDR2, and the Framework3 sequence is located between CDR2 and CDR3.

[0035] Thus, both VL and VH can be visualized approximately as follows, with the CDRs boxed and the N-terminus designated N-. [ka]

[0036] In one embodiment, the antigen binding unit has the following sequence (SEQ ID NO:7) with the three CDRs boxed: [ka] The mouse VH comprises or consists of:

[0037] In other embodiments, the antigen-binding unit comprises a VH comprising or consisting of a sequence having at least 90% or 95% identity to SEQ ID NO:7.

[0038] In one embodiment, the antigen binding unit has the following sequence (SEQ ID NO:8) with the three CDRs boxed: [ka] The mouse VL comprises or consists of:

[0039] In other embodiments, the antigen-binding unit comprises a VL comprising or consisting of a sequence having at least 90% or 95% identity to SEQ ID NO:8.

[0040] VH and VL may be linked by a disulfide bridge or a peptide linker. Alternatively, the two chains may be located within the Fab fragment of the antibody (or any other antigen-binding fragment of the antibody), or within the antibody itself. In one embodiment, the antigen-binding unit comprises or consists of VL-linker-VH (from N-terminus to C-terminus). In another embodiment, the antigen-binding unit comprises or consists of VH-linker-VL (from N-terminus to C-terminus). Such antigen-binding units are often called single-chain Fv fragments (scFv). In order for VH and VL to form a functional antigen-binding unit, the linker must have a certain length. In one embodiment, the linker comprises 10 to 30 amino acid residues. In one embodiment, the linker comprises 15 to 25 amino acid residues, in particular glycine and / or serine residues.

[0041] In one particular embodiment, the linker is a G4S linker, i.e., a peptide linker comprising repeating units having the sequence GGGGS (SEQ ID NO: 21). For example, the linker can be a (G4S)3 (SEQ ID NO: 22), (G4S)4 (SEQ ID NO: 23) or (G4S)5 (SEQ ID NO: 24) linker (i.e., a linker comprising 3, 4 or 5 adjacent repeating G4S units, respectively).

[0042] Alternatively, the linker may be a modified G4S linker comprising one or more amino acid substitutions (optionally conservative amino acid substitutions as defined below) in one or more G4S units (preferably up to one amino acid substitution in one or more G4S units). In particular, the modified G4S units may comprise one or more substitutions of alanine for glycine. One example of a suitable linker, as shown below, has the amino acid sequence set forth in SEQ ID NO: 18, which is a modified (G4S)4 linker in which one glycine residue has been replaced by alanine. GGGGSGGGGSAGGGSGGGGS (SEQ ID NO: 18)

[0043] In an antigen-binding unit, framework sequences can tolerate changes without destroying specificity and affinity for the target antigen. For example, substitution of amino acid residues is better tolerated than deletion or addition of amino acid residues. The replacement of mouse framework sequences with human framework sequences, preferably of similar length, is known as humanization.

[0044] As used herein, the term "conservative amino acid substitution" refers to an amino acid substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain.

[0045] Amino acids with similar side chains tend to have similar properties, so conservative substitution of an amino acid important to the structure or function of a polypeptide can be expected to have less effect on the structure / function of the polypeptide than a non-conservative amino acid substitution at the same position. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine), non-polar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and aromatic side chains (tyrosine, phenylalanine, tryptophan, histidine). Thus, a conservative amino acid substitution can be considered as a substitution in which a particular amino acid residue is replaced with a different amino acid residue of the same family. In particular, products containing conservative amino acid substitutions to a reference sequence are encompassed by this term.

[0046] In one embodiment, each VL and VH herein comprises three CDRs flanked by human framework sequences. Human framework sequences are structurally conserved regions that generally tend to form a β-sheet structure that finely positions the CDRs for specific binding to target antigens under physiological conditions. Many human framework sequences are available from known human antibodies and the International ImMunoGeneTics (IMGT) online database (see Giudicelli et al., Nucleic Acids Research, 2006, Vol. 34, Database Special Issue D781-D784), but the term also encompasses human framework sequences that contain amino acid substitutions. Each of the human framework sequences may optionally contain 0-5 amino acid substitutions relative to the native sequence. An amino acid substitution is a sequence in which an amino acid residue at a particular position is replaced with a different amino acid residue at the corresponding position, as revealed by alignment of the sequences. Each of the human framework sequences may optionally contain one amino acid substitution. Each of the human framework sequences may optionally include two or up to two amino acid substitutions. Each of the human framework sequences may optionally include three or up to three amino acid substitutions. Each of the human framework sequences may optionally include four or up to four amino acid substitutions. Each of the human framework sequences may optionally include five or up to five amino acid substitutions. The substitutions may be conservative substitutions. Even if such framework sequences are not necessarily known from human antibodies, they may pose a lower immunogenicity risk compared to the mouse framework sequences. In one embodiment, 0-5 amino acid residues in the human framework sequences are replaced with the corresponding amino acid residues from the mouse parent sequences found in SEQ ID NOs: 7 and 8.

[0047] Collectively, scFvs that contain CDRs derived from a murine antibody and human framework sequences, each of which may optionally contain 0-5 substitutions, are referred to as humanized scFvs, in some embodiments, some of the substitutions may be back to the parent murine amino acid residues (also known as "backmutations").

[0048] In one embodiment, the human framework sequences are mature human framework sequences available from known human antibodies. Without being bound by theory, such framework sequences may mediate a very low risk of eliciting an unwanted immunogenic response against the antigen-binding unit, while at the same time increasing the likelihood of obtaining a stable binding unit that is well expressed in cell lines.

[0049] Generally, in humanized VH and VL sequences, CDRs are not changed and are kept similar to the parent VH and VL sequences. However, as known in the art, different programs or schemes for determining CDR sequences are available, which may not give exactly the same results in all cases. Thus, different CDR identification schemes may result in different CDR sequences. For example, in the VH or VL sequence, CDR sequences may be shorter or longer, or may be slightly different in position (e.g., in the second scheme, the CDR sequences may be partially moved upstream or downstream relative to the first scheme). Humanization may be performed using a CDR grafting algorithm that uses different versions of the identified CDRs, and these CDRs may be grafted from the original framework onto the selected human sequence. Thus, the humanized sequence may contain CDRs identified according to any of the CDR identification schemes, such as the Kabat scheme, the IMGT scheme, and the Chothia scheme. The corresponding CDR sequences determined by the IMGT and Chothia schemes are presented in Table 2 below. Thus, included herein are humanized VH and VL sequences, and antigen-binding units and binding molecules comprising them, which comprise the CDR sequences presented above or below in Table 2. That is, a VH or VL sequence herein may comprise any of the sets of VH CDR1-3 or VL CDR1-3 presented herein.

[0050] Examples of four different variants of humanized VH are set forth in SEQ ID NOs: 72-75.

[0051] hu-VH1 (SEQ ID NO: 72) EVQIVESGGGLVQPGGSLRLSCATSGFNFNDYFMNWVRQAPGKGLEWIAQIRNKNYNYATYFAESVKGRFTISRDDSKSSVYLQMNSLKTEDTAVYYCTELRYDYWGQGTMVTVSS

[0052] hu-VH2 (SEQ ID NO: 73) EVQIVESGGGLVQPGGSLRLSCATSGFNFNDYFMNWVRQAPGKGLEWVAQIRNKNYNYATYFAESVKGRFTISRDDSKNSVYLQMNSLKTEDTAVYYCTELRYDYWGQGTMVTVSS

[0053] hu-VH3 (SEQ ID NO: 74) QVQIQESGPGLVKPSETLSLTCTTSGFNFNDYFMNWVRQPPGKGLEWIAQIRNKNYNYATYFAESLKSRFTISRDDSKSSVSLKLSSVTAADTAVYYCTELRYDYWGQGTMVTVSS

[0054] hu-VH4 (SEQ ID NO: 75) QVQIQESGPGLVKPSETLSLTCTTSGFNFNDYFMNWIRQPPGKGLEWIAQIRNKNYNYATYFAESLKSRVTISRDDSKNQVSLKLSSVTAADTAVYYCTELRYDYWGQGTMVTVSS

[0055] Examples of four different variants of humanized VL are set forth in SEQ ID NOs: 76-79.

[0056] hu-VL1 (SEQ ID NO: 76) DIVMTQSPDSLAVSLGERATINCKSSQSLLSSGNQKNNLAWYQQKPGQPPKLLIYYASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVADYYCLQHYSSPYTFGGGTKLEIK

[0057] hu-VL2 (SEQ ID NO: 77) DIVMTQSPDSLAVSLGERATINCKSSQSLLSSGNQKNNLAWYQQKPGQPPKLLIYYASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCLQHYSSPYTFGGGTKLEIK

[0058] hu-VL3 (SEQ ID NO: 78) DIVMTQSPLSLPVTPGEPASISCRSSQSLLSSGNQKNNLAWYLQKPGQSPQLLIYYASTRQSGVPDRFSGSGSGTDFTLKISRVEAEDVGDYYCLQHYSSPYTFGGGTKLEIK

[0059] hu-VL4 (SEQ ID NO: 79) DIVMTQSPLSLPVTPGEPASISCRSSQSLLSSGNQKNNLAWYLQKPGQSPQLLIYYASTRQSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQHYSSPYTFGGGTKLEIK

[0060] In the above humanized VH and VL sequences, the CDRs are as determined by the IMGT scheme presented in Table 2 below.

[0061] In one embodiment, the antigen-binding unit comprises the following combination of humanized VH and VL sequences:

[0062] [Table 1]

[0063] More generally, in certain aspects, also provided herein are antigen-binding units, and in particular binding molecules comprising antigen-binding units, that comprise a combination of humanized VH and VL sequences as presented in Table 1.

[0064] In one embodiment, the antigen-binding unit is or comprises an scFv comprising or consisting of the following sequence (SEQ ID NO: 9, CDRs boxed, linker in italics):

[0065] [ka]

[0066] In other embodiments, the antigen-binding unit is or comprises an scFv comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0067] In one embodiment, the antigen-binding unit is or comprises an scFv comprising or consisting of the following sequence (SEQ ID NO: 10, CDRs boxed, linker in italics):

[0068] [ka]

[0069] In another embodiment, the antigen-binding unit is or comprises an scFv comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0070] As can be seen, SEQ ID NO:9 comprises, from N-terminus to C-terminus, a VH of SEQ ID NO:7, a linker of SEQ ID NO:18, and a VL of SEQ ID NO:8, and SEQ ID NO:10 comprises, from N-terminus to C-terminus, a VL of SEQ ID NO:8, a linker of SEQ ID NO:18, and a VH of SEQ ID NO:7.

[0071] A novel chimeric antigen receptor (CAR) is provided. When the CAR of the present invention is expressed on the surface of an immune cell, such an immune cell can be used in medicine. In particular, the immune cell can be used to treat solid tumors expressing p95HER2, which comprises the amino acid sequence set forth in SEQ ID NO: 17. In one embodiment, the immune cell is used to treat p95HER2-positive breast cancer, p95HER2-positive glioma, or other p95HER2-positive cancer.

[0072] As used herein, a CAR is an artificial receptor that includes an extracellular antigen-binding unit, a transmembrane domain, and an intracellular signaling domain. The antigen-binding unit in a CAR is usually an scFv.

[0073] The antigen-binding unit may be directly attached to the transmembrane domain. However, the CAR may include a hinge domain that connects the antigen-binding unit to the transmembrane domain. Thus, the hinge domain may affect the conformation of the antigen-binding unit. As a result, it may affect the ability of the CAR to induce signal transduction in immune cells following binding to the target epitope. If the target epitope is too far from the cell membrane of the target cell, or if the target epitope is otherwise hidden, the immune cell expressing the CAR may not be efficient. Therefore, it is preferable that the target epitope is sufficiently accessible to the immune cell expressing the CAR.

[0074] The transmembrane domain connects the extracellular domain with the intracellular signaling domain. Both the antigen-binding unit and the hinge domain are extracellular domains. That is, when expressed in the cell membrane of an immune cell, they generally face the extracellular environment. As used herein, "transmembrane domain" refers to the part of the CAR that tends to be embedded in the cell membrane when expressed by an immune effector cell. Suitable transmembrane domains are well known to those skilled in the art. In particular, transmembrane domains derived from human proteins CD8α, CD28 or ICOS can be used. The transmembrane domain is believed to transmit a signal into the immune cell upon target binding by the antigen-binding unit.

[0075] "Intracellular signaling domain" refers to the part of the CAR that is located inside the immune cell when the CAR is expressed in the cell membrane. These domains are involved in transmitting signals upon target binding. Various signaling domains are known and can be mixed and matched to match the endogenous signaling mechanisms in immune cells. In one embodiment, the intracellular signaling domain comprises a "signal 1" domain, such as the signaling domains obtained from human proteins CD3zeta, FcR-gamma, CD3epsilon, etc. In general, the "signal 1" domain (e.g., CD3zeta signaling domain) is believed to transmit signals upon antigen binding.

[0076] In other embodiments, the intracellular signaling domain further comprises a costimulatory domain. Such domains are well known and are often referred to as "signal 2" domains, which are believed to transmit signals through costimulatory molecules following the "signal 1" domain. "Signal 2" is important for the maintenance of the signal and survival of the cell. In its absence, as in first generation CARs, CAR-T cells may be efficient in terms of killing and early cytokine release, but frequently become exhausted over time. Thus, the intracellular signaling domain generally comprises both "signal 1" and "signal 2" domains. Examples of such commonly used human "signal 2" domains include the 4-1BB signaling domain, the CD28 signaling domain, and the ICOS signaling domain.

[0077] A CAR in the present disclosure may comprise any of the antigen-binding units described above. For example, a CAR in the present disclosure may comprise an scFv comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10, or a sequence having at least 90% or 95% identity thereto.

[0078] In particular, a CAR in this disclosure may comprise any of the above antigen-binding units in the form of an scFv (e.g., an scFv of SEQ ID NO: 9 or SEQ ID NO: 10) linked to a CD8α hinge. The CD8α hinge is generally the human CD8α hinge of SEQ ID NO: 11, or a variant thereof having at least 90% or 95% sequence identity thereto.

[0079] SDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF (SEQ ID NO: 11)

[0080] In particular, a CAR in the present disclosure may comprise an scFv as defined above (e.g., an scFv of SEQ ID NO: 9 or SEQ ID NO: 10) and an intracellular signaling domain comprising a CD3ζ signaling domain, which is generally the human CD3ζ signaling domain of SEQ ID NO: 14, or a variant thereof having at least 90% or 95% sequence identity thereto.

[0081] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 14)

[0082] In a particular embodiment, the intracellular signaling domain further comprises, in addition to the CD3ζ signaling domain, a costimulatory domain which may be any domain as set out above, but in a particular embodiment is a 41BB costimulatory domain, which is generally the human 41BB costimulatory domain of SEQ ID NO: 13, or a variant thereof having at least 90% or 95% sequence identity thereto.

[0083] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 13)

[0084] The CARs provided herein may in particular comprise a CD8α transmembrane domain, in particular the human CD8α transmembrane domain of SEQ ID NO: 12, or a variant thereof having at least 90% or 95% sequence identity thereto.

[0085] ACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 12)

[0086] In one specific embodiment, the CAR comprises the CD8α hinge and the CD8α transmembrane domain described above.

[0087] In particular, a CAR in the present disclosure may comprise an scFv of SEQ ID NO:9 or SEQ ID NO:10 linked to a CD8α hinge (SEQ ID NO:11), with the proviso that the CAR further comprises a CD8α transmembrane domain (SEQ ID NO:12), and the intracellular signaling domain comprises or consists of a 4-1BB costimulatory domain (SEQ ID NO:13) and a CD3ζ signaling domain (SEQ ID NO:14). Such a CAR may comprise an scFv of SEQ ID NO:15: [ka] It has the amino acid sequence set forth in

[0088] In one particular embodiment, the CAR provided herein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15, or a sequence having at least 90% or 95% identity thereto.

[0089] Sequence identity may be assessed by any convenient method. However, to determine the degree of sequence identity between sequences, computer programs that perform pairwise or multiple alignment of sequences are useful, for example EMBOSS Needle or EMBOSS stretcher (both Rice, P. et al., Trends Genet., 16, (6) pp. 276-277, 2000) may be used for pairwise sequence alignment, and Clustal Omega (Sievers F et al., Mol. Syst. Biol. 7:539, 2011) or MUSCLE (Edgar, RC, Nucleic Acids Res. 32(5):1792-1797, 2004) may be used for multiple sequence alignment. However, other suitable programs may be used. Another suitable alignment program is BLAST, which uses the blastp algorithm for protein alignments and the blastn algorithm for nucleic acid alignments. Whether pairwise or multiple alignments, they should be performed globally (i.e. across the entire reference sequence) rather than locally.

[0090] Sequence alignments and percent identity calculations may be determined, for example, using Clustal Omega standard parameters, i.e., Gonnet matrix, gap opening penalty of 6, gap extension penalty of 1. Alternatively, EMBOSS Needle standard parameters may be used, i.e., BLOSUM62 matrix, gap opening penalty of 10, gap extension penalty of 0.5, or any other suitable parameters may be used.

[0091] The immune cells expressing the CAR herein can be isolated from the patient or a matched donor by leukapheresis or other suitable methods. Such primary cells can be, for example, T cells, NK cells or macrophages. In particular, autologous T cells (both cytotoxic T cells, T helper cells, or a mixture thereof) can be transduced or transfected with a nucleic acid encoding the CAR before a pharmaceutical composition comprising those cells is administered back to the patient. The immune cells expressing the CAR can also be a cell line suitable for clinical use, such as NK-92 cells. Generally, the immune cells expressing the CAR (whether primary cells or cell lines) are T cells (particularly cytotoxic T cells) or NK cells. Of course, when the patient of interest is a human, the preferred cells are human.

[0092] The pharmaceutical composition herein may be a composition suitable for administering therapeutic cells to a patient. The most common route of administration of CAR T cells is intravenous administration. Thus, the pharmaceutical composition may be, for example, a sterile aqueous solution at neutral pH. For example, peripheral blood mononuclear cells of a patient may be obtained by standard leukapheresis. The mononuclear cells may be enriched for T cells before transducing or transfecting with a lentiviral vector or mRNA encoding a CAR. The cells may then be activated with anti-CD3 / CD28 antibody-coated beads. The transduced / transfected T cells may be expanded in cell culture, washed, and formulated into a sterile suspension that can be cryopreserved. In that case, the product is thawed before administration.

[0093] If the tumor is localized, different methods of administration may be used to improve efficacy, for example, local administration of CAR-T cells rather than systemic administration may enhance efficacy.

[0094] The pharmaceutical composition can include a pharma- tically effective dose of the immune cells of the present invention. A pharma- tically effective dose can be, for example, 1×10 immune cells expressing a CAR. 6 ~1×10 10 A pharma- ceutically effective dose can range from, for example, 1 x 10 T cells expressing a CAR. 7 ~1×109 A pharmacologic effective dose can range from, for example, 1×10 NK cells expressing a CAR. 7 ~1×10 9 The range may be 100.

[0095] In order to efficiently express the claimed CAR in immune cells, a conventional leader peptide that promotes placement in the cell membrane can be introduced at the N-terminus. One example of a suitable leader peptide is MESQTQALISLLLWVYGTYG (SEQ ID NO: 16). The leader peptide is likely to be truncated and is unlikely to be present in the functional CAR in the cell membrane.

[0096] Thus, for expression of a second generation CAR, nucleic acids encoding the following can be used: N-LEADER PEPTIDE-VH-LINKER-VL-HINGE-TRANSMEMBRANE DOMAIN-COSTIMULATORY DOMAIN-SIGNALING DOMAIN. Thus, for expression of a second generation CAR, nucleic acids encoding the following may also be used: N-LEADER PEPTIDE-VL-LINKER-VH-HINGE-TRANSMEMBRANE DOMAIN-COSTIMULATORY DOMAIN-SIGNALING DOMAIN

[0097] The nucleic acids encoding the claimed CARs can be in the form of well-known RNA, such as mRNA, or a DNA expression vector.

[0098] Alternatively, the pharmaceutical compositions provided herein may be compositions suitable for administration of the binding molecules (e.g., antibodies) provided herein to patients. Such compositions generally include one or more pharma- ceutically acceptable excipients, etc., known in the art. The binding molecules (e.g., antibodies) provided herein, or pharmaceutical compositions comprising such binding molecules, may be used in medicine / therapy, in particular to treat cancers expressing p95HER2 comprising the amino acid sequence set forth in SEQ ID NO: 17. The binding molecules or pharmaceutical compositions may be used in particular to treat solid cancers, such as breast cancer or glioma.

[0099] In one particular embodiment, a method of treating a p95HER2-positive cancer in a human patient is provided, comprising: a. transducing or transfecting T cells, NK cells, or macrophages with an mRNA encoding any of the CARs herein; and b. repeatedly administering effective doses of a pharmaceutical composition comprising the cells to a patient diagnosed with a p95HER2-positive cancer.

[0100] In one particular embodiment, a method of treating a p95HER2 positive cancer in a human patient is provided, comprising: a. transducing T cells, NK cells, or macrophages with an mRNA encoding a p95HER2 CAR; and b. repeatedly administering effective doses of a pharmaceutical composition comprising said cells to a patient diagnosed with a p95HER2 positive cancer.

[0101] In one particular embodiment, a method of treating a patient diagnosed with breast cancer is provided, comprising: a. obtaining a sample from the patient comprising cancer cells; b. analyzing whether the cancer cells express p95HER2; and c. if the cancer cells are p95HER2 positive, administering a pharmaceutical composition comprising a pharma- ceutical effective dose of T cells, NK cells, or macrophages expressing any of the CARs disclosed herein.

[0102] In one particular embodiment, a method of treating a patient diagnosed with breast cancer is provided, comprising the steps of: a. obtaining a sample from the patient comprising cancer cells; b. analyzing whether the cancer cells express p95HER2; and c. if the cancer cells are p95HER2 positive, administering a pharmaceutical composition comprising a pharma- ceutical effective dose of T cells, NK cells, or macrophages expressing p95HER2 CAR.

[0103] In one particular embodiment, a method of treating a patient diagnosed with breast cancer is provided, comprising the steps of: a. obtaining a sample from the patient comprising cancer cells; b. analyzing whether the cancer cells express p95HER2 by contacting the cells ex vivo with an antibody comprising a VL and a VH as described herein; and c. administering chemotherapy to the patient if the cancer cells are p95HER2 positive.

[0104] As described above, the chemotherapy may be an approved chemotherapy. The chemotherapy may specifically target p95HER2, or cells expressing it.

[0105] In one particular embodiment, a method for diagnosing cancer in a human patient is provided, comprising the steps of: a. obtaining a sample from the patient comprising cells; b. analyzing whether the cells express p95HER2 by contacting the cells ex vivo with a binding molecule provided herein comprising a moiety suitable for detection; and c. diagnosing the patient with cancer if the cells express p95HER2.

[0106] In all the above aspects and embodiments, unless otherwise specified, the CDRs are identified using the Kabat scheme.

[0107] In other embodiments, the CDRs may be identified using the IMGT or Chothia schemes. In particular, the IMGT and Chothia CDR sequences (SEQ ID NOs: 80-91) contained within mouse VH (SEQ ID NO: 7) and mouse VL (SEQ ID NO: 8) are provided in the table below.

[0108] [Table 2]

[0109] In all the above aspects and embodiments, the CDRs may be IMGT or Chothia CDRs, as specified in this aspect. EXAMPLES

[0110] Example 1: Antibody specificity and affinity method

[0111] cell culture Cell lines were cultured in DMEM (Sigma-Aldrich). The medium was supplemented with 100 U / ml penicillin-streptomycin (Sigma-Aldrich) and 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich). Cell lines were incubated at 37°C with 5% CO2 and 100% humidity.

[0112] Preparation of p95HER2 antibody The development of p95HER2 antibody was performed by immunizing rats with cells expressing 611-CTF-HER2. Immunization and hybridoma production were performed by Aldevron (Freiburg, Germany). Briefly, 8- to 12-week-old rats were intradermally injected with 10 μg of immunization vector DNA expressing 611-CTF-HER2 immobilized on gold particles. Selected hybridoma candidates were subcloned by limiting dilution. The collected monoclonal hybridoma supernatants were used for final screening by flow cytometry and / or ELISA.

[0113] Flow cytometry Cells were washed, pelleted, and resuspended in 100 μl FACS buffer (phosphate buffered saline (PBS), pH 7, containing 2% FBS, 2 mM EDTA). The antibody of interest (10 μg / ml) was added and incubated for 30 min in the dark at 4° C. Cells were washed twice and resuspended in 100 μL FACS buffer containing goat anti-rat PE secondary antibody (0.26 μg / ml) and Fixable Viability Dye eFluor™ 780 and incubated for 30 min in the dark at 4° C. Cells were washed and resuspended in 200 μL FACS buffer and analyzed on an LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data was analyzed with FlowJo software (v10.7.1, FlowJo LLC).

[0114] Surface Plasmon Resonance (SPR) SPR was performed as previously described in Gomes & Andreu, Journal of Immunological Methods 259: 217-230, 2002. Briefly, a control anti-HER2 antibody (5 μg / ml) and the antibody of interest (5 μg / ml) were covalently immobilized on the surface of two different flow cells on a sensor chip CM5 (2104988, GE Healthcare) using an amine coupling kit (BR-1000-50, GE Healthcare) and HBS-EP+ buffer. The extracellular domain of p95HER2 peptide (SEQ ID NO: 19) containing a polyhistidine tag at the C-terminus was used as the analyte in serial dilutions from 0.6 to 2500 nM.

[0115] MPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTHHHHHH (SEQ ID NO: 19)

[0116] The kinetics of intermolecular interactions were processed by global curve fitting to a 1:1 bimolecular interaction model. Experiments were performed using a Biacore T200 (GE Healthcare) and all procedures were carried out at 25°C.

[0117] result Generation of p95HER2-specific Abs was performed by immunizing rats with cells transfected with 611-HER2-CTF. For this purpose, HEK-293 cells were transfected with different 611-HER2-CTF constructs and surface expression of p95HER2 was measured using anti-tag antibodies and an irrelevant anti-tag antibody as a control. Immunization of rats to generate mAb hybridomas was performed with cells transfected with pB1-611-CTF-hum.ECD. Initial screening of polyclonal hybridoma culture supernatants (HCS) against p95HER2 was performed using the Intellicyt iQue flow cytometry platform. Based on the mean fluorescence intensity (MFI) values, the top 9 positive clones were selected. HCS from these nine polyclonal hybridomas (p-clones) were then tested for binding to the cell lines p95HER2-T47D, SK-BR-3, T-47D, and SUP-T1 by flow cytometry. HCS from p-clones 1, 2, 3, and 8 were found to bind to p95HER2-T47D, but not to T47D. Only HCS from p-clones 2 and 8 showed binding to SK-BR-3, a cell line expressing full-length HER2. To confirm these results, immunofluorescence (IF) staining was performed with p95HER2-T47D, SK-BR-3, and T-47D. Here, HCS from p-clones 1, 2, and 3 were found to stain p95HER2-T47D, but not T-47D or SK-BR3.

[0118] Based on these data, pClones 1, 2, and 3 were selected for subcloning into monoclonal cultures (mClones) by limiting dilution series. Intellicyt iQue screening of mClones 1, 2, and 3 demonstrated that only mClone 1 bound specifically to transfected cells, mClone 2 bound nonspecifically to nontransfected cells, and mClone 3 was negative. To further test all three mClones, flow cytometric analysis of cell lines p95HER2-T47D, SK-BR-3, and T-47D was performed using HCS. The flow cytometric results confirmed the iQue screening data. Only mClone 1 bound specifically to p95HER2-T47D, while mClone 2 bound to both p95HER2-T47D and SK-BR-3. Furthermore, mClone 1 showed stronger reactivity to p95HER2 compared to pClone 1 as assessed by IF. Based on these screening results, mlon1 was selected for generating a mAb (referred to herein as Oslo-2 antibody).

[0119] Therefore, a monoclonal antibody against p95HER2 was generated in rats using standard hybridoma technology. The reactivity and specificity of the antibody was evaluated by flow cytometry based on binding to a panel of HER2+ / - cell lines derived from different solid tumors or hematological malignancies (Figure 1). Across different breast cancer cell lines, the antibody showed strong reactivity to the p95HER2-T47D cell line, but did not bind to the HER2+ SK-BR-3 and MDA-MB-468 lines, or the HER2- T47D, MCF7, MDA-MB-231 cell lines (Figure 1). Furthermore, the antibody did not bind to the HER2+ lung cancer cell line A549, or the HER2- prostate cancer, pancreatic cancer, lymphoma, and leukemia cell lines (Figure 1). This indicated that the antibody was specific for p95HER2.

[0120] SPR analysis was performed to determine the binding affinity of the antibodies to the p95HER2 peptide of SEQ ID NO: 19 (containing a C-terminal His-tag). The antibodies were immobilized on a chip and the kinetics of the molecular interaction was studied using the extracellular domain of p95HER2 as an analyte at graded concentrations. A control anti-HER2 mAb (ab214275, Abcam, UK), which binds to the cytoplasmic domain of HER2, was used as a reference (Figure 2). The affinity data for the antibodies of interest were corrected by subtraction from the control. As expected, the association (ka) rate increased with increasing p95HER2 peptide concentration. The antibodies of interest showed a low equilibrium dissociation constant (KD=2nM) with the p95HER2 peptide, demonstrating a high affinity interaction with a maximum binding response (Rmax) of 137RU (Figure 2).

[0121] Example 2: Epitope Mapping To identify the specific epitope recognized by the antibody of interest, epitope mapping was performed using synthetic overlapping peptides covering the entire p95HER2 extracellular domain. In the overlapping peptide strategy, sequentially consecutive 15mer peptides overlapping by four amino acids were generated (Figure 3A) and immobilized on a cellulose membrane. The results showed that the antibody bound only to peptide 1, suggesting that the four amino acid sequence MPIW (SEQ ID NO: 33) is essential for binding (Figure 3A&B). This maps the binding epitope to positions 611-614 of full-length HER2 (Figure 3C). The sequence numbers corresponding to the 15mer peptides shown in Figure 3A are presented in the table below.

[0122] [Table 3]

[0123] We next wanted to investigate whether there were additional amino acids involved in antibody binding and to determine whether the binding epitope was continuous or discontinuous. To this end, we selected the region in HER2 from glycine-603 to alanine-622, which spans the N-terminus of the p95HER2 extracellular domain and contains the MPIW 4-mer epitope (SEQ ID NO: 33). We generated peptides containing either N-terminal truncations of this sequence or continuous peptides with two alanine residue substitutions at each position. (Figure 3D). The SEQ ID NOs corresponding to the peptides shown in Figure 3D are provided in the table below.

[0124] [Table 4]

[0125] N-terminal truncation indicated that methionine-611 does not play a significant role in antibody binding, since an initial decrease in binding was only observed after loss of proline-612. Deletion of isoleucine-613 further decreased binding, and removal of tryptophan-614 completely abolished binding (peptide 11) (Figure 3E). The same results as with the substitution of the MPIW (SEQ ID NO: 33) epitope were observed with a double alanine substitution that abolished antibody binding. However, these studies also revealed that substitution of the amino acids immediately adjacent to the original epitope, from lysine-622 to glutamic acid-621, was also important for antibody binding (Figure 3E). Thus, from these studies, we conclude that the binding epitope of the antibody is continuous and has the sequence PIWKFPDEE (SEQ ID NO: 17). The 3D structures of full-length HER2 and p95HER2 indicate that this epitope is hidden in full-length HER2.

[0126] Example 3: Expression levels and in vitro activity of the present invention To generate p95HER2-CAR-T from an antibody, activated T cells were transduced with a CAR construct containing RQR8, signal peptide, p95HER2 scFv (derived from antibody), CD8α hinge, CD8α transmembrane domain, 4-1BB costimulatory domain, and CD3ζ signaling domain in a retroviral expression vector. RQR8 is a compact, epitope-based marker / suicide gene that contains minimal target epitopes derived from CD34 and CD20 antigens. This gene is under the same promoter as the CAR and is separated from the CAR by a self-cleaving protein called 2A. CAR expression was assessed by detecting RQR8 (using an anti-CD34 antibody - QBEND) (Figure 4A). To investigate the functionality of p95HER2-CAR-T in vitro, T cells transduced with the p95HER2-CAR construct and non-transduced T cells were co-cultured with relevant (p95HER2+) and non-relevant (p95HER-) target cells. The cytotoxicity and cytokine production ability of p95HER2-CAR-T were evaluated by different assays. It was observed that p95HER2-CAR-T could induce apoptosis in p95HER2+ target cells (Figure 4B) and secrete the inflammatory cytokines TNFα and IFNγ (Figure 5).

[0127] Example 4: Method for treating model cancer in mice based on the present invention p95HER2-CAR-T (Example 3), which was successfully tested in vitro, was further investigated in vivo. By orthopedically introducing p95HER2-T47D cells into mammary fat pads, our group established a p95HER2-positive orthopedic xenograft mouse model. CAR-T was intravenously injected twice (2 and 5 weeks after tumor implantation) with 5 million cells at each time point. Tumor growth was assessed by in vivo bioluminescence imaging during the treatment time course. Remarkable tumor shrinkage was observed as early as 2 weeks after the first p95HER2-CAR-T injection, and maximum tumor control (tumor disappearance) was observed 5 weeks after the first p95HER2-CAR-T injection (Figure 6). p95HER2-CAR-T was able to persist and grow in vivo for a relatively long period (10 weeks) after the first injection (Figure 7).

[0128] In the figures and examples, "p95HER2-CAR-T" and "p95HER-CAR-T-41BB" both refer to T cells expressing the CAR of SEQ ID NO: 15.

[0129] Example 5: Humanization of antibodies The IMGT VH and VL CDRs of the present invention (SEQ ID NOs: 80-84), presented in Table 2, were grafted to human germline sequences using a CDR grafting algorithm. Table 5 below presents the percentage identity of the parental and humanized sequences to selected human germline sequences.

[0130] [Table 5]

[0131] Sequence trend analysis To ensure that highly undesirable sequence trends were not introduced into the humanized sequences, the parental and humanized sequences were run through Absolute Antibody's sequence trend tool. The sequence trends of greatest concern are glycosylation sites and free cysteines, neither of which are present in the sequences. Deamidation and isomerization motifs are present in the sequences. These modifications are designated high risk and may cause disruptions during manufacturing, but are frequently manageable. If desired, it may be possible to remove these motifs by mutagenesis. Medium and low risk sequence trends also exist, but are rarely reported to cause problems in the antibody manufacturing process.

[0132] Antibody cloning A total of four humanized heavy chains and four humanized light chains were designed (SEQ ID NOs: 72-79). Each of them was synthesized separately and cloned into human IgG1 heavy chain and human kappa light chain expression vectors, respectively. Upon transfection, all possible combinations of humanized sequences were generated, resulting in a total of 16 different humanized antibodies, as presented in Table 1 above.

[0133] Antibody expression and purification Antibodies were expressed and purified by Protein A. The purified proteins were buffer exchanged and concentrated. All antibodies were expressed and all purified products looked as expected under non-reducing and reducing SDS-PAGE.

[0134] Agglutination analysis The purified antibodies were analyzed for aggregation and fragmentation by SEC-HPLC. All purified antibodies showed good monomer content.

Claims

1. A binding molecule comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) that together form an antigen-binding unit, which specifically binds to p95HER2 comprising the amino acid sequence set forth in SEQ ID NO: 17, the VL comprises three complementarity determining regions (CDRs), namely CDR1, CDR2 and CDR3, comprising the amino acid sequences SEQ ID NOs: 1, 2 and 3, respectively; The VH comprises three CDRs, namely CDR1, CDR2 and CDR3, comprising the amino acid sequences SEQ ID NOs: 4, 5 and 6, respectively; binding molecule.

2. the VH comprises the amino acid sequence set forth in SEQ ID NO: 7 or a sequence having at least 90% identity thereto; The binding molecule of claim 1, wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 8 or a sequence having at least 90% identity thereto.

3. The binding molecule of claim 1 , which is an antibody or an antigen-binding fragment thereof.

4. The binding molecule of claim 1 , wherein the antigen-binding unit is an scFv.

5. A chimeric antigen receptor (CAR) comprising the antigen-binding unit of claim 1.

6. The CAR of claim 5, comprising a human CD8α hinge of SEQ ID NO: 11 or a sequence having at least 90% identity thereto.

7. The CAR of claim 6, comprising, from the N-terminus to the C-terminus, a human CD8α hinge, a human CD8α transmembrane domain, a human 4-1BB costimulatory domain, and a human CD3ζ signaling domain.

8. A nucleic acid encoding the binding molecule of claim 1 or the CAR of claim 5.

9. A vector comprising the nucleic acid of claim 8.

10. A cytotoxic immune cell that expresses the CAR according to claim 5 in its cell membrane.

11. The cytotoxic immune cell of claim 10, which is a cytotoxic T cell or an NK cell.

12. A pharmaceutical composition comprising the binding molecule of claim 1. (a) a nucleic acid encoding the binding molecule of claim 1 or the CAR of claim 5, or (b) A pharmaceutical composition comprising a vector containing the nucleic acid of (a).

14. A pharmaceutical composition comprising the cytotoxic immune cells of claim 10.

15. (a) a binding molecule according to claim 1, (b) the cytotoxic immune cell of claim 10; or (c) a nucleic acid encoding the binding molecule of claim 1 or the CAR of claim 5, or a vector comprising said nucleic acid, Use in the manufacture of a medicament for use in the treatment of cancer in a human patient.

16. (a) a binding molecule according to claim 1, (b) the cytotoxic immune cell of claim 10; or (c) comprising a nucleic acid encoding the binding molecule of claim 1 or the CAR of claim 5, or a vector comprising said nucleic acid. A pharmaceutical composition for use in treating cancer that expresses p95HER2, which comprises the amino acid sequence set forth in SEQ ID NO:

17.

17. The pharmaceutical composition of claim 16, wherein the cancer is breast cancer.

18. 1. A method for detecting cancer cells in a sample from a subject, comprising: (a) contacting a sample of cells from the subject with a binding molecule according to any one of claims 1 to 4, wherein the binding molecule further comprises a detection moiety; (b) determining whether the cells express p95HER2; (c) detecting cancer cells in the sample if the cells express p95HER2; A method comprising:

19. A binding molecule comprising a humanized light chain variable domain (VL) and a humanized heavy chain variable domain (VH) that together form an antigen-binding unit, which specifically binds to p95HER2 comprising the amino acid sequence set forth in SEQ ID NO: 17, the VL comprises an amino acid sequence set forth in any one of SEQ ID NOs: 72 to 75; The VH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 76 to 79; binding molecule.

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