Binding molecules targeting the P95 HER2 variant

Abstract

This invention relates to a binding molecule that specifically binds to p95HER2, comprising the amino acid sequence shown in SEQ ID NO:1, including a light chain variable region (VL) and a heavy chain variable region (VH), which together constitute an antigen-binding unit, wherein: (i) the VL comprises three complementarity-determining regions (CDRs) comprising amino acid sequences SEQ ID NO:2, 3, and 4, respectively; and the VH comprises three complementarity-determining regions comprising amino acid sequences SEQ ID NO:5, 6, and 7, respectively; or (ii) the VL comprises three complementarity-determining regions comprising amino acid sequences SEQ ID NO:8, 3, and 9, respectively; and the VH comprises three complementarity-determining regions comprising amino acid sequences SEQ ID NO:5, 6, and 7, respectively. The binding molecule represents a novel targeting unit with practical applications in cancer treatment and diagnosis, and can be provided in the form of a chimeric antigen receptor (CAR).

Description

Technical Field

[0001] This invention relates to the fields of cancer treatment and diagnosis. In particular, this invention relates to novel targeting units and binding molecules, specifically binding proteins or constructs or chimeric antigen receptors (CARs) comprising them, nucleic acids encoding the targeting unit, nucleic acids encoding the binding protein or CAR, immune cells expressing the binding protein or CAR, and their use in cancer treatment. Background Technology

[0002] Many HER2+ breast cancers express HER2 isomers with a truncated CTF (carboxyl terminus), collectively referred to as p95HER2.

[0003] Several antigen-binding units for binding p95HER2 are known, but not all are suitable for CAR. This is illustrated by the failure case described in research publication RD667070, published on October 17, 2019.

[0004] To achieve therapeutic CAR-T cells, these cells need to express sufficient CAR on their cell membranes, and the antigen-binding units must express adequate affinity and specificity. It is foreseeable that only a portion of CAR-T cells that are active in vitro will be able to successfully migrate to the harsh tumor microenvironment of metastatic lesions and / or invasive solid tumors in vivo. Furthermore, CAR-T cells will likely need to maintain their activity long-term to produce therapeutic effects in vivo. Therefore, obtaining novel CARs that can produce therapeutic effects against solid tumors in vivo when expressed on immune cell membranes is not easy, but it is highly desirable.

[0005] Antigen-binding units may also have therapeutic uses in other forms, including as antibodies and antibody-based constructs, such as conjugates (including conjugation with drugs), and as bispecific, trispecific, or other multispecific forms, in which the antigen-binding unit binds to other binding units with different specificities.

[0006] PCT / EP2022 / 079110 describes a novel anti-HER2 antibody capable of specifically binding to p95HER2, referred to herein as the Oslo-2 antibody. This antibody has been shown to be suitable for integration into CARs in scFv form, maintaining sufficient affinity and specificity for p95HER2 in this form. In the clinical application of the Oslo-2 antibody, various variants have been developed and investigated, some of which constitute the subject matter of this invention. Summary of the Invention

[0007] Based on structural modeling and computer-simulated mutation analysis of the Oslo-2 antibody:p95HER2 complex, we generated various mutations in the variable domains of the antibody and studied them in different combinations. This identified several Oslo-2 mutants (or antibody variants) containing mutations in the complementarity-determining region (CDR). Based on further analysis of the mutants and expression optimization in mammalian cells, two specific mutants were selected and successfully expressed.

[0008] Therefore, this paper presents antibody molecules comprising a novel antigen-binding unit based on the variant variable domain sequence described herein. The antigen-binding unit and the binding molecule containing it are capable of specifically binding to cells expressing the highly active 611-CTF isoform of p95HER2 under physiological conditions. The antigen-binding unit described herein exhibits minimal or no binding activity against full-length HER2 under physiological conditions and minimal or no cross-reactivity with healthy tissues. ELISA assays showed that antibodies containing the novel antigen-binding unit possessed similar or comparable binding capacity to p95HER2-derived antigens as parental Oslo-2 antibodies.

[0009] In particular, the novel variant antibody described in this article contains a mutation located in the light chain CDR, and more specifically, it is located in LCDR1, or in both LCDR1 and LCDR3.

[0010] In a first aspect, we provide a binding molecule that specifically binds to p95HER2, comprising the amino acid sequence shown in SEQ ID NO:1. This binding molecule includes a light chain variable region (VL) and a heavy chain variable region (VH), which together form an antigen-binding unit.

[0011] (i) The light chain variable region contains three complementarity-determining regions (CDRs): LCDR1, LCDR2, and LCDR3, which contain amino acid sequences SEQ ID NO: 2, 3, and 4, respectively; and

[0012] The heavy chain variable region contains three CDRs: HCDR1, HCDR2, and HCDR3, which contain amino acid sequences SEQ ID NO: 5, 6, and 7, respectively; or

[0013] (ii) The light chain variable region contains three complementarity-determining regions (CDRs): LCDR1, LCDR2, and LCDR3, which contain amino acid sequences SEQ ID NO: 8, 3, and 9, respectively; and

[0014] The heavy chain variable region contains three CDRs: HCDR1, HCDR2 and HCDR3, which contain amino acid sequences SEQ ID NO:5, 6 and 7, respectively.

[0015] In variant (i) (mutant 4 in the examples below), the heavy chain CDR remains unchanged relative to the parental antibody that produces the binding molecule, while the light chain variable region has an S31W mutation in CDR1 and an H107W mutation in CDR3.

[0016] In variant (ii) (mutant 5 in the examples below), the heavy chain CDR remains unchanged relative to the parental antibody that produces the binding molecule, while the light chain variable region has an S31M mutation in CDR1.

[0017] In one embodiment, the binding molecule may comprise a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 10 or a sequence having at least 80%, 85%, or 90% identity with it, and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 11 or a sequence having at least 80%, 85%, or 90% identity with it.

[0018] In another embodiment, the binding molecule may comprise a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 10 or a sequence having at least 80%, 85%, or 90% identity with it, and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 12 or a sequence having at least 80%, 85%, or 90% identity with it.

[0019] In one embodiment, the binding molecule is in the form of an antibody, which may be a full-length antibody, a fragment thereof, or an antibody derivative, including single-chain antibodies.

[0020] The antigen-binding unit may be a single-chain antibody fragment (scFv).

[0021] In a second aspect, we provide a chimeric antigen receptor (CAR) comprising an antigen-binding unit according to the first aspect. The CAR may comprise a human CD8α hinge region.

[0022] The CAR can be divided into the following regions from the N-terminus to the C-terminus: human CD8α hinge region, human CD8α transmembrane domain, human 4-1BB costimulatory domain and human CD3ζ signal transduction domain.

[0023] In the third aspect, we provide a nucleic acid that encodes either the binding molecule of the first aspect or the CAR of the second aspect.

[0024] Fourthly, we provide a vector containing the nucleic acid from the third aspect.

[0025] In the fifth aspect, we provide an immune cell, particularly a cytotoxic immune cell, which expresses CAR on its cell membrane according to the second aspect.

[0026] In a sixth aspect, we provide a pharmaceutical composition comprising a binding molecule according to the first aspect, a nucleic acid according to the third aspect, a carrier according to the fourth aspect, or an immune cell, particularly a cytotoxic immune cell, according to the fifth aspect.

[0027] In the seventh aspect, we provide a method for treating cancer in a subject (particularly a human subject, or in other words, a human patient), the method comprising the steps of administering immune cells (particularly cytotoxic immune cells) from the fifth aspect or a pharmaceutical composition from the sixth aspect.

[0028] In the eighth aspect, we provide a method for treating cancer in subjects (particularly human patients), the method comprising the following steps:

[0029] (a) Obtaining samples containing cancer cells from a subject (e.g., a patient);

[0030] (b) Analyzing whether cancer cells express p95HER2 by ex vivo contacting cells with a binding molecule according to the first aspect, wherein the binding molecule further comprises a portion suitable for detection;

[0031] (c) If the cancer cells are p95HER2 positive, administer the therapy to the subject (e.g., the patient), especially an approved therapy (e.g., chemotherapy).

[0032] In the ninth aspect, we provide a method for diagnosing cancer, comprising the following steps:

[0033] (a) Obtaining a sample containing cells from a subject (e.g., a human patient);

[0034] (b) Ex vivo contact of cells with a binding molecule according to the first aspect, and analysis of whether the cells express p95HER2, wherein the binding molecule contains a portion suitable for detection; and

[0035] (c) If cells express p95HER2, the subject (e.g., a patient) is diagnosed with cancer.

[0036] In the tenth aspect, we provide a binding molecule according to the first aspect, a CAR according to the second aspect, an immune cell (e.g., cytotoxic immune cells) according to the fifth aspect, or a pharmaceutical composition according to the sixth aspect, for therapeutic use.

[0037] In the eleventh aspect, we provide a binding molecule according to the first aspect, a CAR according to the second aspect, an immune cell (e.g., a cytotoxic immune cell) according to the fifth aspect, or a pharmaceutical composition according to the sixth aspect, for treating cancer wherein the cancer expresses p95HER2.

[0038] In the twelfth aspect, we provide a method for diagnosing cancer in a subject, the method comprising:

[0039] (a) Contacting a cell sample from a subject with a binding molecule as defined herein, wherein the binding molecule further comprises a detection portion;

[0040] (b) Determine whether cells express p95HER2; and

[0041] (c) If cells express p95HER2, the subject (e.g., a patient) is diagnosed with cancer.

[0042] Therefore, the method of the twelfth aspect is an ex vivo method performed on a sample.

[0043] The foregoing aspects of the invention are defined with respect to the binding molecules as defined in the first aspect. However, the disclosure herein extends to other binding molecules that include antigen-binding units of other variants of the variable domain based on the Oslo-2 antibody, as described in the examples below. Therefore, this document also provides for aspects as described above relating to binding molecules, CARs, nucleic acids, vectors, immune cells, pharmaceutical compositions, medical uses, and methods, wherein the binding molecule includes a heavy chain variable region and / or a light chain variable region, and the heavy chain variable region and / or light chain variable region includes a mutated (i.e., variant) sequence as described below. In this regard, reference may be made to the mutants listed in Table 1 below and mutants 1-3 disclosed in the examples below.

[0044] In a specific aspect, this article provides a binding molecule that specifically binds to p95HER2, comprising the amino acid sequence shown in SEQ ID NO: 1. This binding molecule includes a light chain variable region (VL) and a heavy chain variable region (VH), which together form an antigen-binding unit, wherein...

[0045] The light chain variable region contains three complementarity-determining regions (CDRs): LCDR1, LCDR2, and LCDR3, which contain amino acid sequences SEQ ID NO: 16, 3, and 17, respectively (wherein SEQ ID NO: 16 corresponds to residue 31 in the native light chain variable region of antibody Oslo-2, which is S31M / W, and in SEQ ID NO: 17 corresponds to residue 107, which is H107 (native residue) or H107W); and

[0046] The heavy chain variable region contains three CDRs: HCDR1, HCDR2 and HCDR3, which contain amino acid sequences SEQ ID NO:5, 6 and 7, respectively.

[0047] In the above respects, SEQ ID NO: 16 has the following common sequence:

[0048] The residue shown in bold corresponds to residue 31 of the variable region of the natural light chain in SEQ ID NO:15, which is either W or M.

[0049] SEQ ID NO: 17 has the following common sequence:

[0050] The residue shown in bold corresponds to residue 107 of the variable region of the natural light chain in SEQ ID NO:15, which is either H (natural sequence) or W (mutant). Attached Figure Description

[0051] Figure 1 : Expression of novel antibody variants. Figure 1 The SDS-PAGE gel electrophoresis results for two novel antibody variants (variant 4 and variant 5) are shown for quality control.

[0052] Figure 2 Antibody binding ability. Figure 2 The absorbance of the control antibody, Oslo-2 parent antibody, variant 4 and variant 5 antibody at 450 nm wavelength is shown under increasing concentration conditions. Detailed Implementation

[0053] The binding molecule described herein is derived from the antigen-binding domain of the antibody Oslo-2. According to Kabat nomenclature, the CDR of the parental Oslo-2 antibody is disclosed in PCT / EP2022 / 079110 and is shown below:

[0054] VL CDR1 (SEQ ID NO: 13): KSSQSLLSSGNQKNNLA

[0055] VL CDR2 (SEQ ID NO: 3): YASTRQS

[0056] VL CDR3 (SEQ ID NO: 9): LQHYSSPYT

[0057] VH CDR1 (SEQ ID NO: 5): DYFMN

[0058] VH CDR2 (SEQ ID NO: 6): QIRNKNYNYATYFAESLEG

[0059] VH CDR3 (SEQ ID NO: 7): LRYDY

[0060] The amino acid sequence of the VH of the parent antibody is shown in SEQ ID NO: 10, where the three CDR regions are marked with boxes.

[0061]

[0062] The amino acid sequence of the parent antibody VL is shown in SEQ ID NO: 15, where the three CDR regions are marked with boxes.

[0063]

[0064] In the antigen-binding unit of the binding molecule described herein, mutations have been introduced into CDR1 and optional CDR3 of the light chain variable region.

[0065] Specifically, in one variant of the antigen-binding unit of the binding molecule (labeled mutant 4 in this paper), the VH CDR remains unchanged according to Kabat nomenclature, while the VL CDR is as follows:

[0066]

[0067] The S31W mutation in CDR1 and the H107W mutation in VL are shown in bold.

[0068] In another variant of the antigen-binding unit of the binding molecule (labeled mutant 5 in this paper), the VH CDR remains unchanged according to Kabat nomenclature, while the VL CDR is shown below:

[0069]

[0070] The S31M mutation in CDR1 is shown in bold.

[0071] The binding molecules containing novel antigen-binding units described herein typically comprise or consist of one or more proteins (i.e., polypeptide chains) in any suitable form, including antibody forms—this term encompasses all antibody forms and fragments, including, for example, scFv, Fab, immunotoxins, immunoconjugates, bispecific antibodies, CARs, etc.

[0072] Therefore, in one embodiment, the binding molecule provided herein is an antibody or a fragment thereof (i.e., an antigen-binding fragment). Examples of antigen-binding fragments of antibodies include Fab, Fab', and F(ab')2 portions. In another embodiment, the binding molecule is scFv. In yet another embodiment, the binding molecule is CAR.

[0073] In another embodiment, the binding molecule exists in a form conjugated to another portion. More specifically, the conjugate may comprise the binding molecule, or more specifically, may comprise an antigen-binding unit of the binding molecule as defined herein, which is directly or indirectly linked to one or more other portions. This other portion may be a therapeutic or diagnostic agent, or another binding domain, such as a different binding molecule comprising an antigen-binding unit with different specificities. Thus, the binding molecule may be a bispecific, trispecific, or higher-order multispecific binding molecule, such as a bispecific or trispecific antibody. In one embodiment, the binding molecule is a bispecific T-cell connector (BiTE).

[0074] In the conjugate, the binding molecule can be any form of antibody as described herein, such as a full-length antibody or a fragment thereof, or scFv, etc.

[0075] The binding molecules in a conjugate can be directly linked by a bond or indirectly linked by a linker or linker group. The bond can be a peptide bond, and the linker can be a peptide, in which case the conjugate is in the form of a fusion protein. However, in other embodiments, a chemical linker group can be used to link one or more other parts to the binding molecule.

[0076] The binding molecule described herein specifically binds to p95HER2, which contains the amino acid sequence PIWKFPDEE as shown in SEQ ID NO: 1. As further detailed below, SEQ ID NO: 1 is the epitope recognized by the binding molecule described herein.

[0077] Such binding molecules, particularly soluble binding molecules such as antibodies, antigen-binding fragments of antibodies, and scFv, can be used to target cancer cells in their "naked" form (i.e., not conjugated with a second reagent). Alternatively, such binding molecules can carry a toxic payload (e.g., conjugated thereto), such as cytotoxins (e.g., saporin or gelonin) or groups containing radioactive isotopes, such as... 177 Lu、 224 Ra or 225 Ac. The binding molecule coupled with the toxic load can be called an immunotoxin. In this text, "toxin" and "toxic load" refer broadly to any substance (i.e., compound or substance) that has a toxic effect on mammalian cells (especially cytotoxic effects). Therefore, the toxin can be any agent capable of inhibiting the growth and / or survival of mammalian cells (particularly cancer cells). It can be any agent with cytotoxic activity.

[0078] More broadly, the conjugate may comprise a binding molecule (or its antigen-binding unit) linked to a therapeutic agent effective in cancer treatment. It can be any chemotherapeutic agent, and more particularly, any chemotherapeutic agent used or effective in treating any cancer or HER2-positive cancer. The therapeutic agent can be any agent with therapeutic effect; in other words, any drug. Therefore, such conjugates may be referred to as antibody-drug conjugates (ADCs). The use of ADCs in cancer treatment is known in the art. Any known or used drug among the ADCs known or described in the art may be used in the conjugates described herein.

[0079] Examples of representative drugs or cytotoxic agents include: Deschloroclozapine (DCZ), Mertansine (DM1), monomethyl ozonatine E (MMAE), pyrrolobenzodiazepine (PBD), pyrrolobenzodiazepine monoamide (PBD-MA), ozonatine F-hydroxypropionamide (AF-HPA), DM4, Seco-docamycin hydroxybenzamide-azaindole (Seco-DUBA), 7-ethyl-10-hydroxycamptothecin (SN-38), AZI13599185, and AS269 (Amberstatin).

[0080] Furthermore, novel antigen-binding units can be used as diagnostic agents, for example, in the form of naked antibodies or as binding molecules containing detectable markers (such as fluorescent or radioactive portions). The detectable marker can be referred to as a detection moiety or a portion suitable for detection. Such conjugates containing binding molecules or their antigen-binding units linked to a diagnostic agent can be used for in vitro or in vivo diagnostic purposes. Therefore, a detection moiety can be a portion suitable for in vivo detection, such as a tracer or contrast agent used in imaging, such as CT, PET, or SPECT, or an imaging agent, such as a detection moiety detectable by MRI.

[0081] In another embodiment, the binding molecule is in the form of a BiTE. BiTEs are well known and described in the art in the field of cancer therapy. A BiTE can be defined as a bispecific binding molecule comprising the binding molecule of the present invention as defined herein, linked to a second binding molecule capable of specifically binding to T cells. Similarly, bispecific binding molecules capable of binding to other immune cells (e.g., NK cells, macrophages, or monocytes) can be generated. Therefore, the binding molecule can be more generally provided in the form of a bispecific immune cell connector, defined as comprising the binding molecule of the present invention as defined herein, linked to a second binding molecule capable of binding to immune cells. The second binding molecule may comprise an antigen-binding unit derived from an antibody capable of specifically binding to immune cells (e.g., T cells or NK cells). The binding molecules contained in such bispecific binding molecules may be in the form of scFV, linked together by a linking sequence.

[0082] In a typical BiTE, the second binding molecule targets CD3. However, other targets can also be used; for example, the second binding molecule can bind to other cell surface markers specific to T cells or other immune cells, such as NK cell markers. Based on the targets of the second and optional further binding molecules, bispecific or trispecific antibodies can be similarly designed. Multispecific antibodies may or may not contain an Fc region.

[0083] As described in PCT / EP2022 / 079110, the parental Oslo-2 antibody, in its IgG form, has been shown to have a low equilibrium dissociation constant (KD = 2 nM) and a maximum binding response value (Rmax) as high as 137 RU. In one embodiment, the binding molecules described herein exhibit similar orders of magnitude binding in comparable forms. However, this is not an absolute requirement; the binding affinity between the antibody and the binding molecule may vary due to factors such as assay methods, and therefore it is possible that in some assays, the binding molecule may have a lower binding affinity than the Oslo-2 antibody. Reduced binding affinity is acceptable as long as it is sufficient for the binding molecule to effectively bind to its target; in fact, in some cases, reduced affinity can be compensated for by improvements in other parameters, such as improved stability, improved production and development performance, etc. (e.g., increased expression yield in host cells). Such compensation may not be necessary.

[0084] In one implementation, the antigen-binding unit described herein exhibits minimal or no binding activity to full-length HER2 under physiological conditions and minimal or no cross-reactivity to healthy tissues.

[0085] In this study, the target epitope of the antigen-binding unit is considered to be the sequence PIWKFPDEE (SEQ ID NO: 1). The epitope is located in the p95HER2 subtype known as 611-HER2-CTF (SEQ ID NO: 18).

[0086] MPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEA YVMAGVGSPYVSSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIP DLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMG AAKGLQSLPTHDPSPLQRYSEDPTTVPLSSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV (SEQ ID NO: 18)

[0087] Therefore, we provide an antigen-binding unit capable of specifically binding the sequence PIWKFPDEE (SEQ ID NO: 1) under physiological conditions. In one embodiment, the binding molecule (e.g., an antibody) comprises the antigen-binding unit provided herein, having a KD of at least 2 nM.

[0088] As used in this article, an "antigen-binding unit" refers to a structural unit comprising or composed of one or more proteins or portions thereof, capable of binding extracellular target epitopes under physiological conditions. The antigen-binding unit in this article is capable of binding extracellular target epitopes under physiological conditions within a tumor environment. The antigen-binding unit in this article is capable of specifically binding to p95HER2 expressed on cancer cells under physiological conditions. That is, the antigen-binding unit in this article exhibits very little or no binding to full-length HER2 under physiological conditions. Furthermore, the antigen-binding unit in this article exhibits very little or no cross-reactivity with healthy tissues.

[0089] In particular, the antigen-binding units described in this paper are able to bind to epitopes that are masked in intact HER2 but exposed in 611-CTF. This makes them highly specific for the overactive p95HER2 isoform. Specifically, 611-CTF is the only known isoform of p95HER that can broadly induce gene expression associated with metastasis and malignant tumor development.

[0090] Therefore, binding molecules containing the antigen-binding unit provided herein can specifically bind to p95HER2 containing the amino acid sequence shown in SEQ ID NO:1, thereby binding to (or targeting) cancer cells expressing the p95HER2 subtype (such as p95HER2-611-CTF) containing the epitope of SEQ ID NO:1. In particular, the CARs containing such antigen-binding units provided herein enable cytotoxic cells expressing this CAR to target and destroy such cancer cells.

[0091] The antigen-binding unit described herein comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. Such variable domains are well known to those skilled in the art. An antigen-binding unit of an antibody containing both VL and VH is referred to as Fv. An antigen-binding unit as defined herein may comprise a polypeptide chain having both VL and VH sequences (e.g., scFv), or VL and VH may be located on separate polypeptide chains (e.g., Fv).

[0092] Each VL and VH contains three complementarity-determining regions (CDRs) flanked by a frame sequence. The frame sequence can be human, humanized, or mouse. The three VH CDRs of the binding molecule of this invention comprise or consist of SEQ ID NOs: 5, 6, and 7 of the parental antibody as defined above. The three VL CDRs comprise or consist of the following sequences as described above:

[0093] VL CDR1 (SEQ ID NO: 2);

[0094] VL CDR2 (SEQ ID NO: 3);

[0095] VL CDR3 (SEQ ID NO: 4);

[0096] or

[0097] VL CDR1 (SEQ ID NO: 8);

[0098] VL CDR2 (SEQ ID NO: 3);

[0099] VL CDR3 (SEQ ID NO: 9).

[0100] The first frame sequence is located at the N end of CDR1, the second frame sequence is located between CDR1 and CDR2, and the third frame sequence is located between CDR2 and CDR3.

[0101] Therefore, VL and VH can be roughly illustrated as follows, where CDR is marked with a box and the N end is labeled N-:

[0102]

[0103] In one embodiment, the antigen-binding unit comprises a mouse VH, which contains or consists of the following sequences, wherein three CDRs are boxed (SEQ ID NO: 10):

[0104]

[0105] In another embodiment, the antigen-binding unit comprises a VH, which comprises or consists of a sequence having at least 80%, 85%, 90%, or 95% identity with SEQ ID NO: 10.

[0106] In one embodiment, the antigen-binding unit comprises a VL, wherein the VH comprises or consists of the following sequences, wherein three CDRs are boxed (SEQ ID NO: 11):

[0107]

[0108] In another embodiment, the antigen-binding unit comprises a VL, wherein the VH comprises or consists of a sequence having at least 80%, 85%, 90%, or 95% identity with SEQ ID NO: 11.

[0109] In one embodiment, the antigen-binding unit comprises a VL, wherein the VH comprises or consists of the following sequences, wherein three CDRs are indicated by boxes (SEQ ID NO: 12):

[0110]

[0111] In another embodiment, the antigen-binding unit comprises a VL, wherein the VH comprises or consists of a sequence having at least 80%, 85%, 90%, or 95% identity with SEQ ID NO: 12.

[0112] In SEQ ID NO: 11 and 12, compared to the parental antibody VL sequence shown in SEQ ID NO: 15, the VL sequence of the new variant also contains an L-to-I substitution (L112I) at the penultimate residue. The parental Oslo-2 antibody is a rat IgG2a type antibody, and the VL sequence of the new variant is modified to achieve reassortment to the mouse IgG1 form.

[0113] VH and VL can be linked via a disulfide bridge or a peptide linker. Alternatively, the two chains can 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 commonly referred to as single-chain Fv fragments (scFv). The linker must be of a certain length to allow VH and VL to form a functional antigen-binding unit. In one embodiment, the linker contains 10 to 30 amino acid residues. In another embodiment, the linker contains 15 to 25 amino acid residues, particularly glycine and / or serine residues.

[0114] In one particular embodiment, the linker is a G4S linker, i.e., a peptide linker containing a repeating unit of the sequence GGGGS (SEQ ID NO: 19). For example, the linker may be a (G4S)3 (SEQ ID NO: 20), (G4S)4 (SEQ ID NO: 21), or (G4S)5 (SEQ ID NO: 22) linker (i.e., a linker containing 3, 4, or 5 adjacent repeating G4S units, respectively).

[0115] The linker can also be a modified G4S linker containing one or more amino acid substitutions (optionally conserved amino acid substitutions, as defined below) in one or more G4S units, preferably having at most one amino acid substitution in one or more G4S units. In particular, the modified G4S unit may contain one or more glycine residues replaced by alanine. As shown below, a suitable linker example has the amino acid sequence shown in SEQ ID NO: 23, which is a modified (G4S)4 linker in which one glycine residue is replaced by alanine.

[0116] GGGGSGGGGSAGGGSGGGGS (SEQ ID NO: 23)

[0117] In antigen-binding units, the frame sequence can allow for a degree of variation without affecting specificity and affinity for the target antigen. For example, substitution of amino acid residues may be more acceptable than deletion or addition of amino acid residues. The process of replacing a mouse frame sequence with a human frame sequence, preferably of similar length, is called humanization. This document includes variants of SEQ ID NO: 10, 11, and 12 that have at least 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 10, 11, or 12, and whose frame regions contain one or more amino acid modifications (i.e., the CDR sequence is unmodified). In particular, this includes variants with humanized frame regions.

[0118] As used herein, the term “conservative amino acid substitution” refers to an amino acid substitution in which one amino acid residue is replaced by another amino acid residue having a similar side chain.

[0119] Amino acids with similar side chains generally have similar properties. Therefore, a conserved substitution of an amino acid that is crucial to the structure or function of a polypeptide can be expected to have a smaller impact on the polypeptide's structure or function compared to a non-conserved 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), nonpolar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, a conserved amino acid substitution can be considered as replacing a specific amino acid residue with a different amino acid residue from the same family. In particular, products containing conserved amino acid substitutions relative to a reference sequence fall within the scope of this term.

[0120] In one implementation, each VL and VH herein contains three CDRs surrounded by a human frame sequence. The human frame sequence is a structurally conserved region that typically tends to form a β-sheet structure, which, under physiological conditions, precisely positions the CDRs to achieve specific binding to the target antigen. Many human frame sequences are available from known human antibodies and the International Immunogenetic Information System (IMGT) online database (see Giudicelli et al, Nucleic AcidsResearch, 2006, Vol. 34, Database issue D781–D784), but the term also encompasses human frame sequences containing amino acid substitutions. Each human frame sequence may optionally contain 0 to 5 amino acid substitutions relative to the native sequence. An amino acid substitution is a sequence change in which an amino acid residue at a specific position is replaced by a different amino acid residue at the corresponding position during sequence alignment. Each human frame sequence may optionally contain 1 amino acid substitution. Each human frame sequence may optionally contain 2 or more than 2 amino acid substitutions. Each human frame sequence may optionally contain 3 or more than 3 amino acid substitutions. Each human frame sequence may optionally contain 4 or more than 4 amino acid substitutions. Each human frame sequence may optionally contain five or more amino acid substitutions. These substitutions may be conserved. Even if such frame sequences are not necessarily derived from known human antibodies, they may still pose a lower risk of immunogenicity compared to mouse frame sequences. In one embodiment, 0 to 5 amino acid residues in the human frame sequence are replaced with the corresponding amino acid residues from the mouse parental sequences in SEQ ID NO: 10 and 15.

[0121] A single-chain antibody fragment comprising a CDR from a mouse antibody and a human framework sequence, wherein each framework sequence may optionally contain 0 to 5 amino acid substitutions, collectively referred to as a humanized single-chain antibody fragment. In some embodiments, partial substitutions may be restored to parental mouse amino acid residues (also known as "reverse mutations").

[0122] In one implementation, the human framework sequence is a mature human framework sequence available from known human antibodies. Without being limited to theoretical possibilities, such framework sequences may have a very low risk of inducing an undesirable immune response against the antigen-binding unit, while increasing the likelihood of obtaining a binding unit stably expressed in cellular systems.

[0123] Generally, in humanized VH and VL sequences, the CDR remains unchanged and is preserved as in parental VH and VL sequences. However, as is known in the art, different procedures or schemes exist for determining CDR sequences, and these methods do not always yield completely consistent results. Therefore, different CDR identification schemes may produce different CDR sequences. For example, they may be shorter or longer, or their positions within the VH or VL sequences may differ slightly (e.g., in the second scheme, the CDR sequence may be partially shifted upstream or downstream relative to the first scheme). Humanization can be performed using CDR transfer algorithms, which utilize different variants of the identified CDR to transfer the CDR from the original frame to a selected human sequence. Thus, humanized sequences can contain CDRs identified according to any CDR identification scheme, such as the Kabat scheme, the IMGT scheme, and the Chothia scheme.

[0124] In some representative embodiments, the antigen-binding unit is or comprises an scFv, which contains or consists of the following sequences from the N-terminus to the C-terminus:

[0125] (i) The VH sequence of SEQ ID NO:10 or a sequence having at least 80% sequence identity with it, the linker sequence of SEQ ID NO:23, and the VL sequence of SEQ ID NO:11 or a sequence having at least 80% sequence identity with it; or

[0126] (ii) The VH sequence of SEQ ID NO:10 or a sequence having at least 80% sequence identity with it, the linker sequence of SEQ ID NO:23, and the VL sequence of SEQ ID NO:12 or a sequence having at least 80% sequence identity with it; or

[0127] (iii) The VL sequence of SEQ ID NO:11 or a sequence having at least 80% sequence identity with it, the linker sequence of SEQ ID NO:23, and the VH sequence of SEQ ID NO:10 or a sequence having at least 80% sequence identity with it; or

[0128] (iv) The VL sequence of SEQ ID NO:12 or a sequence having at least 80% sequence identity with it, the linker sequence of SEQ ID NO:23, and the VH sequence of SEQ ID NO:10 or a sequence having at least 80% sequence identity with it.

[0129] As described above, antibodies can take many forms, including antibody fragments. All of these forms are included. An antibody may contain one or more antigen-binding domains, or one or more VH regions and / or one or more VL regions. The VH and / or VL regions may be contained within a single chain (peptide) or located on different chains (peptides). Therefore, an antibody may contain one or more polypeptide chains, such as two or four polypeptide chains. A single polypeptide may contain one or more VH regions and / or one or more VL regions, such as containing both VH and VL regions simultaneously.

[0130] Therefore, the antigen-binding domain may contain one or more polypeptides (chains), each polypeptide containing one or more VH regions and / or one or more VL regions.

[0131] Furthermore, a polypeptide (chain) containing a variable region sequence may contain all or part of a constant region sequence. For example, in the case of VH, it may contain one, two, or all three of the heavy chain constant regions CH1, CH2, and CH3; in the case of VL, it may contain all or part of the light chain constant region (CL).

[0132] Therefore, the term "antibody" includes all known forms of antibodies, including full-length or complete antibodies, any antigen-binding fragment thereof, single-chain or single-chain derivatives thereof, and synthetic or artificial antibody constructs comprising at least one VH region as defined herein and at least one VL region as defined herein, as well as polymers thereof, such as dimers, trimers, or higher-order polymeric antibodies. It should be understood that the term includes recombinant and engineered antibodies.

[0133] More broadly, the term "antibody" can be considered as any binding protein containing an antigen-binding domain (specifically, an antigen-binding domain derived from an antibody), which may be called an immune-binding protein. Therefore, the term "antibody" is used to refer to any antibody-like molecule having an antigen-binding region obtained from or derived from an antibody. This antibody or a fragment thereof contains at least one VH region and at least one VL region.

[0134] In one embodiment, the antibody is an immunoglobulin antibody, more specifically an antibody or fragment thereof comprising at least two heavy chains and at least two light chains.

[0135] In any form herein, when an antibody contains constant regions, the heavy chain contains the VH region and all or part of the heavy chain constant region as defined herein, and the light chain contains the VL region and all or part of the light chain constant region as defined herein. When an antibody contains complete constant regions of both the heavy and light chains, it is referred to as a complete antibody or full-length antibody. Such full-length / complete antibodies represent a preferred embodiment.

[0136] Based on the type of heavy chain domain, antibodies are classified into five major classes: IgA, IgD, IgE, IgG, and IgM. All of these classes are included, but IgA and IgG, especially IgG, are preferred. Some of these are further subdivided into subclasses or isotypes, such as IgG1, IgG2, IgG3, and IgG4. For example, camel antibodies are IgG antibodies that often possess the IgG2 or IgG3 constant domain. All subclasses are included here. The constant heavy chain domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known. Suitable constant heavy chain regions are known and available in the art.

[0137] The light chains of mammalian antibodies can be divided into two distinct types: κ and λ, and either one can be used. Similarly, suitable light chain constant region sequences are known and available in the art.

[0138] In one embodiment, the heavy chain constant region is or contains all or part of the mouse IgG1 constant region having an amino acid sequence as shown in SEQ ID NO: 24 or an amino acid sequence that is at least 90% sequence identical thereto.

[0139] SEQ ID NO: 24, Mouse IgG1 constant region

[0140] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLMISLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTKPREEQINSTFRSSVSELPILHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0141] In another embodiment, the light chain constant region is or contains all or part of the mouse κ light chain constant region having an amino acid sequence as shown in SEQ ID NO: 25 or an amino acid sequence that is at least 90% identical to it.

[0142] SEQ ID NO: 25, mouse κ light chain constant region

[0143] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0144] Alternatively, in another embodiment, the heavy chain constant region is or contains all or part of the human IgG1 constant region having an amino acid sequence as shown in SEQ ID NO: 37 or an amino acid sequence that is at least 90% identical to it.

[0145] SEQ ID NO: 37, Human IgG1 constant region

[0146] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0147] In another embodiment, the light chain constant region is or contains all or part of the human κ light chain constant region having an amino acid sequence as shown in SEQ ID NO: 38 or an amino acid sequence that is at least 90% identical to it.

[0148] SEQ ID NO: 38, human κ light chain constant region

[0149] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0150] When binding molecules are used in a soluble form, i.e., not expressed on the cell surface (e.g., as antibodies and certain conjugates), they can be conveniently produced by expression in host cells (e.g., cell lines used for production). Such cell lines are conveniently mammalian cells, such as HEK293 cells.

[0151] Techniques and materials for such expression are well known and widely available in the art. For example, nucleotide sequences encoding variable domains of the binding molecule as defined herein can be designed and codon-optimized for expression in mammalian cells. Sequences can be subcloned into cloning and expression vectors. For example, in antibody production, vectors containing sequences for expressing desired isotypes and subtypes of antibodies (e.g., sequences encoding desired or selected constant regions) are available or known in the art. The heavy and light chains can be expressed using different vectors, or the binding molecule can be expressed using a single vector, depending on factors such as the chosen antibody form.

[0152] The binding molecule or its individual polypeptide chain can be expressed in the form of a signal peptide. A variety of different signal sequences are known and described in the art.

[0153] For example, for the expression of VH and VL peptides in this paper, the signal peptide shown in SEQ ID NO: 35 can be used.

[0154] SEQ ID NO: 35

[0155] MPLLLLLPLLWAGALA

[0156] A novel chimeric antigen receptor (CAR) is provided. When the CAR described herein is expressed on the surface of immune cells, such immune cells can be used medically. Specifically, the immune cells can be used to treat solid tumors expressing p95HER2, wherein p95HER2 comprises the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, the immune cells are used to treat p95HER2-positive breast cancer, p95HER2-positive glioma, or other p95HER2-positive cancers, such as bladder cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, lung cancer, kidney cancer, and colorectal cancer.

[0157] As used in this article, a CAR is an artificial receptor that includes an extracellular antigen-binding unit, a transmembrane domain, and an intracellular signal transduction domain. The antigen-binding unit in a CAR is typically an scFv.

[0158] The antigen-binding unit can be directly linked to the transmembrane domain. However, the CAR may include a hinge domain connecting the antigen-binding unit to the transmembrane domain. Therefore, the hinge domain may affect the stereoconformity of the antigen-binding unit. This, in turn, may affect the CAR's ability to bind to the target epitope and subsequently trigger signal transduction into the immune cell. If the target epitope is too far from the target cell membrane, or if the target epitope is hidden, the CAR-expressing immune cell may not function effectively. Therefore, it is preferable that the target epitope has sufficient accessibility to the CAR-expressing immune cell.

[0159] Transmembrane domains connect extracellular domains to intracellular signaling domains. Both the antigen-binding unit and the hinge domain are extracellular domains, meaning they typically face the extracellular environment when expressed on the cell membrane of immune cells. As used herein, "transmembrane domain" refers to a portion of the CAR that, when expressed by immune effector cells, tends to embed itself within the cell membrane. 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. Transmembrane domains are thought to transmit signals into the immune cell after the antigen-binding unit binds to its target.

[0160] "Intracellular signal transduction domains" refer to the portion of the CAR located inside the immune cell when it is expressed on the cell membrane. These domains participate in signal transduction after binding to the target. A variety of signal transduction domains are known to exist, which can be combined and customized to suit endogenous signal transduction mechanisms in immune cells. In one implementation, the intracellular signal transduction domain includes a "signal 1" domain, such as a signal transduction domain obtainable from human proteins like CD3ζ, FcR-γ, and CD3ε. It is generally believed that "signal 1" domains (e.g., the CD3ζ signal transduction domain) transmit signals upon antigen binding.

[0161] In another implementation, the intracellular signal transduction domain further includes a co-stimulatory domain. Such domains are well-known and often referred to as "signal 2" domains, and are believed to transmit signals via co-stimulatory molecules following the "signal 1" domain. "Signal 2" is crucial for signal maintenance and cell survival. Without it, as demonstrated by first-generation CARs, CAR-T cells may be highly efficient in killing and early cytokine release, but often become depleted over time. Therefore, intracellular signal transduction domains typically include both "signal 1" and "signal 2" domains. Common examples of human "signal 2" domains include the 4-1BB signal transduction domain, the CD28 signal transduction domain, and the ICOS signal transduction domain.

[0162] The CAR of this disclosure may comprise any antigen-binding unit as described above. For example, the CAR of this disclosure may comprise the scFv as described above.

[0163] In particular, the CAR of this disclosure may comprise any of the aforementioned antigen-binding units in the form of an scFv attached to the CD8α hinge region. The CD8α hinge region is typically the human CD8α hinge region as shown in SEQ ID NO: 26, or a variant having at least 90% or 95% sequence identity with it.

[0164] SDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF (SEQ ID NO: 26)

[0165] Specifically, the CAR in this disclosure may include the scFv as defined above and an intracellular signaling domain containing a CD3ζ signaling domain. The CD3ζ signaling domain is typically the human CD3ζ signaling domain shown in SEQ ID NO: 27, or a variant thereof having at least 90% or 95% sequence identity with it.

[0166] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 27)

[0167] In one particular embodiment, in addition to the CD3ζ signaling domain, the intracellular signaling domain also includes a co-stimulatory domain, which can be any of the such domains described above, but in one particular embodiment is a 4-1BB co-stimulatory domain. The 4-1BB co-stimulatory domain is typically the human 4-1BB co-stimulatory domain as shown in SEQ ID NO: 28, or a variant having at least 90% or 95% sequence identity with it.

[0168] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 28)

[0169] The CARs provided herein may in particular include a CD8α transmembrane domain, especially the human CD8α transmembrane domain as shown in SEQ ID NO: 29, or a variant thereof having at least 90% or 95% sequence identity with it.

[0170] ACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 29)

[0171] In one particular implementation, the CAR includes the CD8α hinge region and the CD8α transmembrane domain as described above.

[0172] Specifically, the CAR of this disclosure may include the scFv as described above, which is connected to the CD8α hinge region (SEQ ID NO: 26), wherein the CAR further includes a CD8α transmembrane domain (SEQ ID NO: 29), and wherein the intracellular signal transduction domain includes or is composed of a 4-1BB co-stimulatory domain (SEQ ID NO: 28) and a CD3ζ signal transduction domain (SEQ ID NO: 27).

[0173] Sequence identity can be assessed by any convenient method. However, computer programs for pairwise or multiple sequence alignment are very useful when determining the degree of sequence identity between sequences. For example, EMBOSS Needle or EMBOSSstretcher (both from Rice, P. et al., Trends Genet., 16, (6) pp276-277, 2000) can be used for pairwise sequence alignment, while Clustal Omega (Sievers F et al., Mol. Syst. Biol. 7:539, 2011) or MUSCLE (Edgar, RC, Nucleic Acids Res. 32(5):1792-1797, 2004) can be used for multiple sequence alignment, but any other suitable program may also be used. Another suitable alignment program is BLAST, where the blastp algorithm is used for protein alignment and the blastn algorithm is used for nucleic acid alignment. Whether pairwise or multiple alignment, it must be performed globally (i.e., across the entire region of the reference sequence), rather than locally.

[0174] Sequence alignment and identity calculation can be performed using, for example, the standard Clustal Omega parameters: matrix Gonnet, gap opening penalty of 6, gap extension penalty of 1. Alternatively, the standard EMBOSS Needle parameters can be used: matrix BLOSUM62, gap opening penalty of 10, gap extension penalty of 0.5. Any other suitable parameters may also be used.

[0175] The CAR-expressing immune cells described in this article can be obtained from patients or compatible donors through leukoablation or other suitable methods. For example, such primary cells can be T cells, NK cells, or macrophages. Specifically, autologous T cells (including cytotoxic T cells, helper T cells, or mixtures thereof) can be transduced or transfected with CAR-encoding nucleic acids before the drug composition containing these cells is reinfused into the patient. CAR-expressing immune cells can also be clinically suitable cell lines, such as NK-92 cells. Typically, CAR-expressing immune cells (whether primary cells or cell lines) are T cells (especially cytotoxic T cells) or NK cells. Of course, when the intended patient is human, human cells are preferred.

[0176] The pharmaceutical compositions described herein may be compositions suitable for administering therapeutic cells to subjects (e.g., patients). The most common route of administration for CAR-T cells is intravenous administration. Therefore, the pharmaceutical compositions may be, for example, sterile aqueous solutions with a neutral pH. Peripheral blood mononuclear cells from a subject can be obtained, for example, via a standard leukocyte separation procedure. The mononuclear cells may be T-cell enriched prior to transduction or transfection using a lentiviral vector or mRNA encoding the CAR. Subsequently, the cells may be activated using magnetic beads coated with anti-CD3 / CD28 antibodies. The transduced / transfected T cells may be expanded in cell culture medium, washed, and formulated into a sterile suspension for cryopreservation. If so, the product should be thawed before administration.

[0177] When the tumor is in a localized state, different administration methods can be used to enhance the therapeutic effect. For example, regional or local administration of CAR-T cells, rather than systemic administration, may improve the therapeutic effect.

[0178] The pharmaceutical composition may contain a pharmaceutically effective dose of the immune cells described herein. For example, a pharmaceutically effective dose may be 1 × 10⁻⁶. 6 Up to 1×10 10 Within the range of immune cells expressing CAR. For example, a pharmaceutically effective dose can be 1 × 10⁻⁶. 7 Up to 1×10 9 Within the range of CAR-expressing T cells. For example, a pharmaceutically effective dose can be 1 × 10⁻⁶. 7 Up to 1 × 10⁹ NK cells expressing CAR.

[0179] To achieve efficient expression of the desired protected CAR in immune cells, a conventional leader peptide can be introduced at its N-terminus to facilitate its localization on the cell membrane. An example of a suitable leader peptide is MESQTQALISLLLWVYGTYG (SEQ ID NO:14). It is generally believed that the leader peptide will be cleaved and is unlikely to be present in functional CARs on the cell membrane.

[0180] Therefore, to express second-generation CARs, nucleic acids encoding the following components can be used:

[0181] N-lead peptide-VH-linker-VL-hinge region-transmembrane domain-co-stimulatory domain-signal transduction domain. Therefore, to express second-generation CARs, nucleic acids encoding the following components can also be used:

[0182] N-lead peptide-VL-linker-VH-hinge region-transmembrane domain-co-stimulatory domain-signal transduction domain.

[0183] The nucleic acid encoding the CAR that requires protection can be in the form of well-known RNA, such as mRNA, or a DNA expression vector.

[0184] The pharmaceutical compositions provided herein can also be compositions suitable for administering the conjugating molecules (e.g., antibodies) provided herein to a subject (e.g., a patient). Such compositions typically comprise one or more pharmaceutically acceptable excipients or analogues known in the art. The conjugating molecules (e.g., antibodies) provided herein, or pharmaceutical compositions comprising such conjugating molecules, can be used for medical / therapeutic purposes, particularly for treating cancers expressing p95HER2 containing the amino acid sequence shown in SEQ ID NO: 1. The conjugating molecules or pharmaceutical compositions are particularly useful for treating solid tumors, such as breast cancer or glioma.

[0185] Subjects can be any human or non-human animal, particularly any mammal, including domesticated, farm, livestock, sporting, or zoo animals. In particular, subjects are humans, and therefore the methods and uses described herein are specifically applicable to human patients.

[0186] In one particular implementation, a method for treating p95HER2-positive cancer in a subject (e.g., a human patient) is provided, comprising the following steps:

[0187] a. Transduce or transfect T cells, NK cells, or macrophages using mRNA encoding any of the CARs described in this article;

[0188] b. Repeatedly administer an effective dose of the pharmaceutical composition containing the cells to a subject (e.g., a patient) diagnosed with p95HER2-positive cancer.

[0189] In one particular implementation, a method for treating p95HER2-positive cancer in a subject (e.g., a human patient) is provided, comprising the following steps:

[0190] a. Transducing T cells, NK cells, or macrophages using mRNA encoding p95HER2 CAR;

[0191] b. Repeatedly administer an effective dose of the pharmaceutical composition containing the cells to a subject (e.g., a patient) diagnosed with p95HER2-positive cancer.

[0192] In one particular implementation, a method is provided for treating a subject diagnosed with breast cancer (e.g., a patient), wherein the method includes the following steps:

[0193] a. Obtaining samples containing cancer cells from a subject (e.g., a patient);

[0194] b. Analyze whether the cancer cells express p95HER2; and

[0195] c. If the cancer cells are p95HER2 positive, administer a pharmaceutical composition containing a pharmaceutically effective dose of any of the CARs disclosed herein expressing T cells, NK cells, or macrophages.

[0196] In one particular implementation, a method is provided for treating a subject diagnosed with breast cancer (e.g., a patient), wherein the method includes the following steps:

[0197] a. Obtaining samples containing cancer cells from a subject (e.g., a patient);

[0198] b. Analyze whether the cancer cells express p95HER2; and

[0199] c. If the cancer cells are p95HER2 positive, administer a drug composition containing a pharmaceutically effective dose of p95HER2 CAR-expressing T cells, NK cells, or macrophages.

[0200] In one particular implementation, a method is provided for treating a subject diagnosed with breast cancer (e.g., a patient), wherein the method includes the following steps:

[0201] a. Obtaining samples containing cancer cells from a subject (e.g., a patient);

[0202] b. Expose cells to antibodies containing VL and VH as described herein under in vitro conditions to analyze whether the cancer cells express p95HER2; and

[0203] c. If the cancer cells are p95HER2 positive, administer cancer therapy (e.g., chemotherapy) to the subject (e.g., the patient).

[0204] As mentioned above, chemotherapy can be an approved chemotherapy regimen. This chemotherapy may specifically target p95HER2 or cells that express p95HER2.

[0205] In one particular implementation, a method for diagnosing cancer in a subject (e.g., a human patient) is provided, wherein the method includes the following steps:

[0206] a. Obtaining a sample containing cells from a subject (e.g., a patient);

[0207] b. In vitro contact the cells with the binding molecule provided herein and analyze whether the cells express p95HER2, wherein the binding molecule contains a detectable portion; and

[0208] c. If cells express p95HER2, the subject (e.g., a patient) is diagnosed with cancer.

[0209] In all the foregoing aspects and embodiments, unless otherwise specified, CDRs are identified using the Kabat scheme, as described above. However, as noted in other aspects and embodiments above, the binding molecule may comprise an antigen-binding unit containing a variant VH and / or VL sequence comprising any combination of mutations specified in Table 1 below. The above disclosure of details of the invention is similarly applicable to such aspects and embodiments.

[0210] Specifically, the binding molecule may contain an antigen-binding unit, wherein VH and VL are defined as follows:

[0211] (i) The VL contains three complementarity-determining regions (CDRs): LCDR1, LCDR2, and LCDR3, which contain amino acid sequences SEQ ID NO: 2, 3, and 4, respectively; and

[0212] VH contains three CDRs: HCDR1, HCDR2, and HCDR3, which contain amino acid sequences SEQ ID NO: 5, 6, and 30, respectively. (where SEQ ID NO: 30 contains the mutation L107W), referred to herein as mutant 1; or

[0213] (ii) VL contains three complementarity-determining regions (CDRs): LCDR1, LCDR2, and LCDR3, each containing the amino acid sequence SEQ ID NO: 31 ( (where SEQ ID NO: 31 contains the mutations S31W and N38M), SEQ ID NO: 3 and SEQ ID NO: 32 ( (where SEQ ID NO: 32 contains the mutations Y108D and S109W); and

[0214] VH contains three CDRs: HCDR1, HCDR2, and HCDR3, containing amino acid sequences SEQ ID NO; 5, 6, and 30, respectively, referred to in this paper as mutant 2; or

[0215] (iii) The VL contains three complementarity-determining regions (CDRs): LCDR1, LCDR2, and LCDR3, containing amino acid sequences SEQ ID NO: 8, 3, and 9, respectively; and

[0216] VH contains three CDRs: HCDR1, HCDR2 and HCDR3, which contain amino acid sequences SEQ ID NO: 5, 6 and 30, respectively, and are referred to as mutant 3 in this paper.

[0217] In addition to the mutated VL sequence, mutants 1, 2, and 3 also contain a mutation (L017W) in VH (specifically in CDR3). Mutant 1 contains the same VL mutation in CDR1 and CDR3 as mutant 4 described in this paper. Mutant 3 contains the same VL mutation in CDR1 as mutant 5 described in this paper. Compared to mutant 4, mutant 2 contains an additional VL mutation in CDR1 and a substitution mutation in CDR3.

[0218] In one embodiment, the binding molecule may comprise a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 33 or a sequence having at least 80%, 85%, or 90% identity with it, and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 11 or a sequence having at least 80%, 85%, or 90% identity with it (corresponding to mutant 1).

[0219] In another embodiment, the binding molecule may comprise a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 33 or a sequence having at least 80%, 85%, or 90% identity with it, and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 34 or a sequence having at least 80%, 85%, or 90% identity with it (corresponding to mutant 2).

[0220] In another embodiment, the binding molecule may comprise a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 33 or a sequence having at least 80%, 85%, or 90% identity with it, and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 12 or a sequence having at least 80%, 85%, or 90% identity with it (corresponding to mutant 3).

[0221] SEQ ID NO: 33 – VH mutant containing L107W

[0222]

[0223] SEQ ID NO: 34 – VL mutant containing S31W, N38M, Y108D and S109W

[0224]

[0225] Example

[0226] Example 1: Antibody Design

[0227] method:

[0228] In the first step, the structure of the Oslo-2 antibody-p95HER2 complex was determined through docking studies. Ten docking structures were generated using information-driven docking technology, and a common structure was identified based on epitope overlap. FoldX analysis also showed a favorable interaction between the antibody and the p95HER2 antigen (-10.87 kcal / mol). Subsequently, computer simulations were used to optimize the antibody's binding affinity and exploitability.

[0229] This includes mutation analysis of the complementary site of the Oslo-2 antibody and computational mutation analysis of the complementary site residues, aiming to discover: 1) residues important for binding; and 2) mutations that may improve binding. This method identified multiple point mutations, and subsequently identified a significant point mutation (<-1.5 kcal / mol) that significantly improves binding affinity with high confidence.

[0230] At six mutation sites, a total of 32 mutations resulted in interaction energies of less than -1.5 kcal / mol, leading to 35,279 antibody variants. Most of these mutations were located in the light chain region of the antibodies.

[0231] As expected, we observed a normal distribution of mutations. We focused on the leftmost outliers on the numerical scale, which significantly improved the interaction energy. However, it is equally important to note that increasing the number of mutation sites is likely to significantly improve binding. However, more mutations may also lead to CDR conformational instability and loss of binding ability. Therefore, the goal is to find the optimal interaction energy while minimizing the number of mutations.

[0232] Further research on the top 5 mutations with different numbers of mutation sites and mutation frequencies will enable us to identify key high-frequency mutations.

[0233] The feasibility of antibody development was then assessed using the Therapeutic Antibody Analyzer (TAP). Table 1 below lists the analyzed mutations. We found that "Surface Hydrophobic Plaque (PSH)" is the most critical parameter, which needs to be optimized based on the antibody variant. Mutants were identified as having lower PSH scores.

[0234] Table 1

[0235]

[0236] Subsequently, we conducted computer simulation aggregation analysis to identify mutations that could reduce the aggregation ability of easily aggregated regions (APRs) in antibodies.

[0237] result:

[0238] Through computer simulation analysis, five antibody mutants were identified, referred to as mutants 1 to 5 in this paper. The VH and VL sequences of mutants 1-5 (also known as variants 1 to 5) are shown below.

[0239] A. In heavy and light chains:

[0240] 1. H:L107W / L:S31W / L:H107W: Excellent performance in all exploitability parameters and APR.

[0241]

[0242] 2. H:L107W / L:S31W / L:N38M / L:Y108D / L:S109W: Although the PSH score is slightly higher than the amber marker range in the TAP method, it shows minimal aggregation in the ANuPP spectrum.

[0243]

[0244] 3. H:L107W / L:S31M: Performance is optimal when the number of mutations is minimal.

[0245]

[0246] B. Only in light chains:

[0247] 4. L:S31W / L:H107W: Performs well in all exploitability parameters and APR.

[0248] 5. L:S31M: Performance is optimal when the number of mutations is minimal.

[0249]

[0250] Based on the initial findings, mutants 4 and 5 were selected for further research.

[0251] Example 3: Antibody Production

[0252] summary:

[0253] Prior to synthesis, the variable domains of novel Oslo-2 monoclonal antibody variant sequences (mutants 4 and 5) were designed and optimized for expression in mammalian cells (HEK293). Subsequently, the sequences were subcloned into suitable isotypes and expression vectors for immunoglobulin heavy and light chains (IgG1). HEK293 cells were passaged to the optimal stage for transient transfection. After transient transfection with heavy and light chain expression vectors, cells were cultured for 6–14 days. 80 ml volumes of cells were transfected to obtain approximately 0.5–1.0 mg of antibody. Cultures were collected and purified in one step using affinity chromatography. Antibody concentrations were determined by UV spectroscopy. The yields of all antibodies were determined, and aggregation levels were determined by SEC-HPLC.

[0254] method:

[0255] Gene Synthesis and Cloning

[0256] The variable heavy and light chain domains were designed with appropriate restriction sites at their 5' and 3' ends for cloning into the Absolute Antibody cloning and expression vector. The variable domain sequences were codon-optimized for expression in human cells. After gene synthesis, the variable domains were cloned into the appropriate species and type of Absolute Antibody vector. The correct sequences were verified by Sanger sequencing, and the raw data were analyzed using DNASTAR Lasergene software. Following confirmation, plasmid DNA of appropriate size was prepared to obtain a sufficient quantity of high-quality DNA for transfection.

[0257] Expression and purification

[0258] HEK 293 (human embryonic kidney 293) mammalian cells were passaged to the optimal stage for transient transfection. Cells were transiently transfected with heavy and light chain expression vectors and cultured for another 6 days. Cultures were collected by centrifugation at 4000 rpm and filtered through a 0.22 µM filter. The first step of purification was Protein A affinity chromatography, eluting with pH 3.0 citrate buffer followed by neutralization with 0.5 M Tris (pH 9.0). The eluted protein was then transferred to PBS via a desalting column. Antibody concentration was determined by UV spectroscopy, and the antibody was concentrated as needed.

[0259] Antibody analysis

[0260] Antibody purity was determined by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and HPLC (high performance liquid chromatography). SEC-HPLC was performed using an Agilent 1100 series instrument with a size-appropriate size SEC column. Antibody expression titers were determined by Protein A HPLC.

[0261] result:

[0262] Figure 1 Quality control results for the expression of novel antibodies, referred to as mutant 4 and mutant 5 (also known as variant 4 and variant 5), are shown. 80 ml of transfected cell culture was collected and purified by one-step affinity chromatography. For quality control, the purified antibodies were subjected to electrophoresis on an SDS-PAGE gel.

[0263] Example 3: Evaluation of the binding ability of novel antibody variants using enzyme-linked immunosorbent assay (ELISA)

[0264] method :

[0265] Indirect ELISA

[0266] Antibody binding was determined by indirect ELISA.

[0267] At a concentration of 2.5 µg / mL, microplates (protein sequence: MPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTHHHHHH) (SEQ ID NO: 36) were coated with 50 µL of antigen and incubated for 1 hour with shaking at 300 rpm. Then, 200 µL of PBS solution containing 1% casein was added, and the plates were blocked at 4°C for 16 hours. Finally, the microplates were washed twice with PBS containing 0.05% Tween-20. In separate offline microplates, antibody test samples were prepared using a 3-fold serial dilution method with a starting concentration of 3.0 µg / mL, and double-replicated assays were performed. 50 µL of each test sample was transferred to the test plate and incubated for 1 hour with shaking at 300 rpm. The plates were washed four times with PBS containing 0.05% Tween-20. Add 50 µL of horseradish peroxidase (HRP)-labeled anti-human IgG antibody (catalog number A8792, Sigma) diluted 1:4000 to each well and incubate with shaking at 300 rpm for 1 hour. Wash the plate four times with PBS containing 0.05% Tween-20, followed by two washes with water. Add 100 µL of tetramethylbenzidine (TMB) substrate (catalog number 10076433, Fisher) and incubate for 10 minutes. Stop the reaction by adding 50 µL of 1M HCl and measure the absorbance at 450 nm using a Byonoy microplate reader.

[0268] result:

[0269] Both variant 4 and variant 5 exhibited the ability to bind to the antigen, and the absorbance readings of variant 4 and variant 5 were higher than those of the parental Oslo-2 monoclonal antibody. Figure 2 ).

Claims

1. A binding molecule that specifically binds to p95HER2, comprising the amino acid sequence shown in SEQ ID NO:

1. It contains a light chain variable region (VL) and a heavy chain variable region (VH), which together constitute the antigen-binding unit, wherein: (i) The VL comprises three complementarity-determining regions (CDRs): LCDR1, LCDR2, and LCDR3, which respectively contain amino acid sequences SEQ ID NO: 2, 3, and 4; and The VH contains three CDRs: HCDR1, HCDR2, and HCDR3, which respectively contain amino acid sequences SEQ ID NO: 5, 6, and 7; or (ii) The VL comprises three complementarity-determining regions (CDRs): LCDR1, LCDR2, and LCDR3, which respectively contain amino acid sequences SEQ ID NO: 8, 3, and 9; and The VH contains three CDRs: HCDR1, HCDR2 and HCDR3, which contain amino acid sequences SEQ ID: 5, 6 and 7, respectively.

2. The binding molecule as claimed in claim 1, wherein: (i) The VH comprises an amino acid sequence as shown in SEQ ID NO: 10, or a sequence having at least 90% identity with it, and The VL contains an amino acid sequence as shown in SEQ ID NO: 11, or a sequence having at least 90% identity with it; or (ii) The VH comprises an amino acid sequence as shown in SEQ ID NO: 10, or a sequence having at least 90% identity with it, and The VL contains an amino acid sequence as shown in SEQ ID NO: 12, or a sequence having at least 90% identity with it.

3. The binding molecule as described in claim 1 or 2, wherein the molecule is an antibody or a fragment thereof.

4. The binding molecule as claimed in claim 1 or 2, wherein the antigen-binding unit is scFv.

5. The binding molecule according to any one of claims 1 to 4, wherein the molecule is in a form conjugated with a cytotoxic agent.

6. The binding molecule according to any one of claims 1 to 4, wherein the molecule is a bispecific or trispecific antibody.

7. The binding molecule of claim 6, wherein the molecule is a bispecific T-cell connector (BiTE).

8. A chimeric antigen receptor (CAR) comprising an antigen-binding unit as described in any one of claims 1, 2 or 4.

9. The CAR of claim 7, comprising the human CD8α hinge region of SEQ ID NO: 26 or a sequence having at least 90% identity with it.

10. The CAR of claim 9, wherein from the N-terminus to the C-terminus it comprises a human CD8α hinge region, a human CD8α transmembrane domain, a human 4-1BB co-stimulatory domain, and a human CD3ζ signal transduction domain.

11. A nucleic acid encoding a binding molecule as described in any one of claims 1 to 7 or a CAR as described in any one of claims 8 to 10.

12. A vector comprising the nucleic acid as described in claim 11.

13. A cytotoxic immune cell that expresses CAR on its cell membrane as described in any one of claims 8 to 10.

14. The cytotoxic immune cell of claim 13, wherein the cell is a cytotoxic T cell or an NK cell.

15. A pharmaceutical composition comprising the binding molecule as described in any one of claims 1 to 7.

16. A pharmaceutical composition comprising the nucleic acid as claimed in claim 11 or the carrier as claimed in claim 12.

17. A pharmaceutical composition comprising cytotoxic immune cells as described in claim 13 or 14.

18. A method of treating cancer in a human patient, comprising the step of administering a binding molecule as described in any one of claims 1 to 7, cytotoxic immune cells as described in claim 13 or 14, or a pharmaceutical composition as described in any one of claims 15 to 17.

19. A method for treating cancer in a human patient, comprising the steps of: a. Obtain a sample containing cancer cells from the patient; b. Analyzing whether the cancer cells express p95HER2 by ex vivo contacting cells with the binding molecule as described in any one of claims 1 to 7, wherein the binding molecule further comprises a detectable portion; and c. If the cancer cells are p95HER2 positive, then administer chemotherapy to the patient.

20. A method for diagnosing cancer, comprising the steps of: a. Obtaining cell-containing samples from human patients; b. Analyzing whether the cells express p95HER2 by ex vivo contacting the cells with the binding molecule as described in any one of claims 1 to 7, wherein the protein contains a detectable portion; and c. If the cells express p95HER2, the patient is diagnosed with cancer.

21. A conjugating molecule as described in any one of claims 1 to 7, a CAR as described in any one of claims 8 to 7 or 10, a cytotoxic immune cell as described in claim 13 or 14, or a pharmaceutical composition as described in any one of claims 15 to 17 for therapeutic purposes.

22. A binding molecule as described in any one of claims 1 to 7, a chimeric antigen receptor as described in any one of claims 8 to 10, a cytotoxic immune cell as described in claim 13 or 14, or a pharmaceutical composition as described in any one of claims 15 to 17 for treating cancer, wherein the cancer expression comprises p95HER2 with the amino acid sequence shown in SEQ ID NO:

1.

23. The combination molecule, CAR, cytotoxic immune cell or pharmaceutical composition used as described in claim 22, wherein the cancer is breast cancer.

24. A method for diagnosing cancer in a subject, the method comprising: (a) Contacting a cell sample from the subject with a binding molecule as described in any one of claims 1 to 7, wherein the binding molecule further comprises a detection portion; (b) Determine whether the cells express p95HER2; and (c) If the cells express p95HER2, the patient is diagnosed with cancer.

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