WO2022228579A1 - Récepteurs antigéniques chimériques ciblant gpc3 et leurs procédés d'utilisation - Google Patents

Récepteurs antigéniques chimériques ciblant gpc3 et leurs procédés d'utilisation Download PDF

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WO2022228579A1
WO2022228579A1 PCT/CN2022/090894 CN2022090894W WO2022228579A1 WO 2022228579 A1 WO2022228579 A1 WO 2022228579A1 CN 2022090894 W CN2022090894 W CN 2022090894W WO 2022228579 A1 WO2022228579 A1 WO 2022228579A1
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amino acid
seq
cells
acid sequence
cdrs
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Qingling JIANG
Yafeng Zhang
Shu Wu
Shuai Yang
Xiaojie TU
Miaomiao SHI
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Nanjing Legend Biotech Co., Ltd.
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This disclosure relates to single-domain antibodies targeting GPC3, chimeric antigen receptors targeting GPC3, and methods of use thereof.
  • liver cancers are the fourth most common cause of cancer-related death and rank sixth in terms of incident cases.
  • the World Health Organization estimates that more than 1 million patients will die from liver cancer in 2030.
  • Half of all liver cancer cases and deaths are estimated to occur in China (Chen W.Q. et al., CA Cancer J Clin. Mar-Apr 2016; 66 (2) : 115-32) .
  • Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancers.
  • HCC Hepatocellular carcinoma
  • Most of HCC cases are diagnosed at advanced-stage that aren’t suitable for curative treatment, such as surgical resection, liver transplantation, radiofrequency ablation or trans-arterial chemoembolization (Villanueva A.
  • CAR T cell therapy has been re-energized by the application of chimeric antigen receptor (CAR) T cell therapy in cancers.
  • CAR T cells are engineered to express synthetic receptors, which have a modular design with the following components: an antigen-binding domain, a hinge, a transmembrane region and an intracellular signaling domain (Rafiq S. et al., Nat Rev Clin Oncol. 2020 Mar; 17 (3) : 147-167) .
  • the extracellular antigen-binding domain may comprise a single chain variable fragment (scFv) .
  • the CARs Upon binding to the target tumor antigen, the CARs can activate the T cells to launch specific anti-tumor response in a major histocompatibility complexes (MHC) -independent manner.
  • MHC major histocompatibility complexes
  • Anti-GPC3 antibodies have been used for liver cancer detection. Although antibody-dependent cellular cytotoxicity (ADCC) and the complement-dependent cytotoxicity (CDC) research programs have been reported, no clinical use of anti-GPC3 antibodies have been approved. Only the GC33 antibody, codrituzumab (PCT Application No. PCT/JP2005/013103) entered clinical research. In Phase II clinical trial, codrituzumab was not found to be effective against liver cancer (Abou-Alfa G.K. et al., J Hepatol. 2016 Aug; 65 (2) : 289-95) . Thus, there is a need in the field to further develop new therapies targeting GPC3 for solid tumors.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the disclosure relates to anti-GPC3 CAR-T cell therapy for the treatment of cancer patients with GPC3-positive cancer, including e.g., liver cancer.
  • Genetically engineered T cells can recognize and attack target cells. These T cells can be isolated from the host and genetically modified using e.g., suitable virus mediated or non-viral means of transfection. Thereafter, the modified T cells can be infused back into the patients as adoptive cell therapy.
  • the single domain antibodies (sdAbs) disclosed herein can provide additional advantages. They have a much smaller size than that of conventional antibodies (e.g., IgG) and are more stable. They can bind to hidden epitopes that are not accessible by conventional antibodies (e.g., IgG) , which makes them suitable as the extracellular antigen-binding domain of the CARs expressed by CAR-T cells.
  • the disclosure is related to a chimeric antigen receptor comprising: (a) a heavy chain single variable domain (V H H) that specifically binds to Glypican 3 (GPC3) ; (b) a transmembrane region; and (c) an intracellular signaling domain, in some embodiments, the heavy chain single variable domain (V H H) comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the V H H CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected V H H CDR1 amino acid sequence, the V H H CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected V H H CDR2 amino acid sequence, and the V H H CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected V H H CDR3 amino acid sequence.
  • the selected VHH CDRs 1, 2, and 3 amino acid sequences are one of the following:
  • V H H CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 2, 4, and 6, respectively;
  • V H H CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 9, 11, and 13, respectively;
  • V H H CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 16, 18, and 20, respectively;
  • V H H CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 23, 25, and 27, respectively;
  • V H H CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 30, 32, and 34, respectively;
  • V H H CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 37, 39, and 41, respectively;
  • V H H CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 44, 46, and 48, respectively;
  • V H H CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 51, 53, and 55, respectively;
  • V H H CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 58, 60, and 62, respectively.
  • the V H H consists of or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 64, 65, 66, 67, 68, 69, 70, 71, or 72.
  • the V H H comprises an amino acid sequence that is identical to SEQ ID NO: 64, 65, 66, 67, 68, 69, 70, 71, or 72.
  • antigen-binding domain further comprises a second heavy chain single variable domain (V H H2) .
  • the V H H2 comprises complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the V H H2 CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected V H H2 CDR1 amino acid sequence, the V H H2 CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected V H H2 CDR2 amino acid sequence, and the V H H2 CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected V H H2 CDR3 amino acid sequence.
  • the selected V H H2 CDRs 1, 2, and 3 amino acid sequences are one of the following:
  • V H H2 CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 2, 4, and 6, respectively;
  • V H H2 CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 9, 11, and 13, respectively;
  • V H H2 CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 16, 18, and 20, respectively;
  • V H H2 CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 23, 25, and 27, respectively;
  • V H H2 CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 30, 32, and 34, respectively;
  • V H H2 CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 37, 39, and 41, respectively;
  • V H H2 CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 44, 46, and 48, respectively;
  • V H H2 CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 51, 53, and 55, respectively;
  • V H H2 CDRs 1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 58, 60, and 62, respectively.
  • the V H H2 consists of or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%sequence identity to SEQ ID NO: 64, 65, 66, 67, 68, 69, 70, 71, or 72.
  • the V H H2 comprises an amino acid sequence that is identical to SEQ ID NO: 64, 65, 66, 67, 68, 69, 70, 71, or 72.
  • the two V H Hs are joined by a linker.
  • the linker comprises the amino acid sequence of GGGGS (SEQ ID NO: 99) .
  • the linker comprises the amino acid sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 100) .
  • the antigen-binding domain is connected to the transmembrane region (or domain) by a hinge region (or domain) .
  • the hinge region comprises an IgG-based hinge, or a membrane-proximal region from CD8 and/or CD28, or a portion thereof.
  • the hinge region comprises a CD8 membrane-proximal region.
  • the hinge region comprises an amino acid sequence set forth in SEQ ID NO: 106, or an amino acid sequence that is at least 90%identical to SEQ ID NO: 106.
  • the hinge region comprises a CD28 membrane-proximal region.
  • the hinge region comprises an amino acid sequence set forth in SEQ ID NO: 108, or an amino acid sequence that is at least 90%identical to SEQ ID NO: 108.
  • the transmembrane region comprises a transmembrane region of CD4, CD8, and/or CD28, or a portion thereof. In some embodiments, the transmembrane region comprises a transmembrane region of CD8. In some embodiments, the transmembrane region comprises an amino acid sequence set forth in SEQ ID NO: 107, or an amino acid sequence that is at least 90%identical to SEQ ID NO: 107. In some embodiments, the transmembrane region comprises a transmembrane region of CD28. In some embodiments, the transmembrane region comprises an amino acid sequence set forth in SEQ ID NO: 109, or an amino acid sequence that is at least 90%identical to SEQ ID NO: 109.
  • the intracellular signaling domain comprises a primary intracellular signaling sequence of an immune effector cell. In some embodiments, the intracellular signaling domain is or comprises a functional signaling domain of CD3 zeta. In some embodiments, the intracellular signaling domain is or comprises the amino acid sequence set forth in SEQ ID NO: 103, or an amino acid sequence that is at least 90%sequence identical to SEQ ID NO: 103.
  • the intracellular signaling domain further comprises a costimulatory signaling domain.
  • the costimulatory signaling domain comprises a functional signaling domain from a protein selected from the group consisting of a MHC class I molecule, a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein) , an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1, CD11a/CD18, 4-1BB (CD137) , B7-H3, CDS, ICAM-1, ICOS (CD278) , GITR, BAFFR, LIGHT, HVEM (LIGHTR) , KIRDS2, SLAMF7, NKp80 (KLRF1) , NKp44, NKp30,
  • the costimulatory signaling domain is or comprises a functional signaling domain from OX40, CD28, 4-1BB, and/or ICOS, or a signaling portion thereof. In some embodiments, the costimulatory signaling domain comprises an intracellular signaling domain of 4-1BB and/or CD28. In some embodiments, the costimulatory signaling domain is or comprises an amino acid sequence set forth in SEQ ID NO: 102, 110, or 111; or an amino acid sequence that is at least 90%identical to SEQ ID NO: 102, 110, or 111.
  • the disclosure is related to a chimeric antigen receptor consisting of or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identity to SEQ ID NO: 82, 83, 84, 85, 86, 87, 88, 89, or 90.
  • the amino acid sequence is identical to SEQ ID NO: 82, 83, 84, 85, 86, 87, 88, 89, or 90.
  • the disclosure is related to a chimeric antigen receptor consisting or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identity to SEQ ID NO: 91, 92, 93, 94, 95, 96, 97, or 98.
  • the amino acid sequence is identical to SEQ ID NO: 91, 92, 93, 94, 95, 96, 97, or 98.
  • the disclosure is related to a polynucleotide encoding the chimeric antigen receptor as described herein.
  • the disclosure is related to a polynucleotide encoding two or more of the chimeric antigen receptors as described herein.
  • the sequences encoding the two or more chimeric antigen receptors are joined by a sequence encoding a self-cleavage peptide.
  • the self-cleavage peptide is P2A or T2A peptide.
  • the polynucleotide as described herein further comprises a sequence encoding a signal peptide and/or a tag.
  • the signal peptide is or comprises an amino acid sequence set forth in SEQ ID NO: 104; or an amino acid sequence that is at least 90%identical to SEQ ID NO: 104.
  • the tag is FLAG tag (DYKDDDDK; SEQ ID NO: 105) .
  • the disclosure is related to a vector comprising the polynucleotide as described herein.
  • the vector is a viral vector.
  • the viral vector is a retroviral vector or a lentiviral vector.
  • the disclosure is related to an engineered cell expressing the chimeric antigen receptor as described herein.
  • the disclosure is related to an engineered cell, comprising the polynucleotide as described herein or the vector as described herein.
  • the engineered cell is a primary cell obtained from a subject (e.g., a human patient) . In some embodiments, the engineered cell is a cell line.
  • the engineered cell is an immune cell.
  • the immune cell is an NK cell or a T cell.
  • the engineered cell is a T cell.
  • the T cell is CD8+.
  • the T cell is CD4+.
  • the T cell is ⁇ -T cell.
  • the T cell is isolated from a human patient.
  • the engineered cell expresses two or more different chimeric antigen receptors.
  • the disclosure is related to a method for producing the engineered cell, comprising introducing a vector as described herein into a cell in vitro or ex vivo.
  • the vector is a viral vector and the introducing is carried out by transduction.
  • the disclosure is related to a method of generating a population of cells, comprising introducing a nucleic acid into a cell, wherein the nucleic acid comprises the polynucleotide as described herein, or a nucleic acid encoding the chimeric antigen receptor as described herein.
  • the disclosure is related to a pharmaceutical composition, comprising the engineered cell as described herein and a pharmaceutically acceptable carrier.
  • the disclosure is related to a method of treating a GPC3-associated disease or disorder in a subject, comprising administering the engineered cell or therapeutically effective amount of the pharmaceutical composition as described herein to the subject.
  • the GPC3-associated disease or disorder is a cancer.
  • the cancer is liver cancer.
  • the disclosure is related to an anti-GPC3 antibody or antigen-binding fragment thereof comprising: a heavy chain single variable domain (V H H) comprising complementarity determining regions (CDRs) 1, 2, and 3, in some embodiments, the V H H CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected V H H CDR1 amino acid sequence, the V H H CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected V H H CDR2 amino acid sequence, and the V H H CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected V H H CDR3 amino acid sequence.
  • the selected V H H CDRs 1, 2, and 3 amino acid sequences are one of the following:
  • V H H CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 2, 4, and 6, respectively;
  • V H H CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 9, 11, and 13, respectively;
  • V H H CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 18, and 20, respectively;
  • V H H CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 23, 25, and 27, respectively;
  • V H H CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 30, 32, and 34, respectively;
  • V H H CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 37, 39, and 41, respectively;
  • V H H CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 44, 46, and 48, respectively;
  • V H H CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 51, 53, and 55, respectively;
  • V H H CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 58, 60, and 62, respectively.
  • the disclosure is related to an antibody or antigen-binding fragment thereof that binds to GPC3 comprising a heavy chain single variable domain (V H H) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a selected V H H sequence, in some embodiments, the selected V H H sequence is selected from SEQ ID NOs: 64, 65, 66, 67, 68, 69, 70, 71, or 72.
  • V H H heavy chain single variable domain
  • the antibody or antigen-binding fragment specifically binds to human GPC3. In some embodiments, the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is a multi-specific antibody (e.g., a bispecific antibody) .
  • the disclosure is related to an antibody or antigen-binding fragment thereof comprising the V H H CDRs 1, 2, and 3 of the antibody or antigen-binding fragment thereof as described herein.
  • the antibody or antigen-binding fragment thereof comprises a IgG1, IgG2, IgG3, or IgG4 Fc.
  • the disclosure is related to a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof as described herein.
  • the disclosure is related to a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen binding fragment thereof as described herein and a pharmaceutically acceptable carrier.
  • the disclosure is related to a method of treating a subject having cancer, comprising administering an effective amount of the antibody or antigen-binding fragment thereof or therapeutically effective amount of the pharmaceutical composition as described herein to the subject.
  • antibody refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope in an antigen.
  • CDR complementary determining region
  • Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies) , single-chain antibodies, single variable domain (V H H) antibodies, chimeric antibodies, human antibodies, and humanized antibodies.
  • an antibody can contain an Fc region of a human antibody.
  • the term antibody also includes derivatives, e.g., multi-specific antibodies, bi-specific antibodies, single-chain antibodies, diabodies, and linear antibodies formed from these antibodies or antibody fragments.
  • the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen.
  • the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain, a variable domain of light chain or a V H H) .
  • variable domains include, e.g., Fab, Fab’, F (ab’) 2 , Fv fragments, scFv, and V H H.
  • the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present disclosure is provided.
  • Veterinary and non-veterinary applications are contemplated in the present disclosure.
  • Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) .
  • patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates.
  • non-human primates e.g., monkey, chimpanzee, gorilla, and the like
  • rodents e.g., rats, mice, gerbils, hamsters, ferrets, rabbits
  • lagomorphs e.g., swine (e.g., pig, miniature pig)
  • equine canine, feline, bovine, and other domestic, farm, and zoo animals.
  • the phrases “specifically binding” and “specifically binds” mean that the antibody or an antigen-binding fragment interacts with its target molecule preferably to other molecules, because the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general.
  • An antibody that specifically binds to the target molecule may be referred to as a target-specific antibody.
  • an antibody that specifically binds to GPC3 may be referred to as a GPC3-specific antibody or an anti-GPC3 antibody.
  • bispecific antibody refers to an antibody that binds to two different epitopes.
  • the epitopes can be on the same antigen or on different antigens.
  • trispecific antibody refers to an antibody that binds to three different epitopes.
  • the epitopes can be on the same antigen or on different antigens.
  • multispecific antibody refers to an antibody that binds to two or more different epitopes.
  • the epitopes can be on the same antigen or on different antigens.
  • a multispecific antibody can be e.g., a bispecific antibody or a trispecific antibody.
  • the multispecific antibody binds to two, three, four, five, or six different epitopes.
  • V H H refers to the variable domain of a heavy chain only antibody.
  • the V H H is a humanized V H H.
  • a “chimeric antigen receptor” or “CAR” refers to a fusion protein comprising an extracellular domain capable of binding to an antigen, and an intracellular region comprising one or more intracellular signaling domains derived from signal transducing proteins. These intracellular signaling domains are typically different from the polypeptide from which the extracellular domain is derived.
  • the extracellular domain can be any proteinaceous molecule or part thereof that can specifically bind to a predetermined antigen.
  • the extracellular domain comprises an antibody or antigen binding fragment thereof.
  • the intracellular signaling domain can be any oligopeptide or polypeptide domain known to function to transmit a signal causing activation or inhibition of a biological process in a cell, for example, activation of an immune cell such as a T cell or a NK cell.
  • tandem CAR refers to a CAR comprising two or more extracellular domain capable of binding to an antigen.
  • a tandem CAR can have 2, 3, 4, 5, 6, 7, 8, 9, or 10 extracellular domains that are capable of binding to an antigen.
  • These antigen-binding domains can be the same or different. In some embodiments, they can bind to the same or different antigens. In some embodiments, they can bind to different epitopes on the same antigen.
  • FIG. 1 shows immune response of pre-immune serum and post-immune serum after final boost against human GPC3.
  • Absorbance at 450 nm indicates the level of immune response determined by ELISA.
  • FIG. 2 shows immune response of conventional heterotetrameric antibodies (IgG1) and heavy-chain only antibodies (IgG2 and IgG3) in post-immune serum after boosts against human GPC3. Corresponding immunoglobulin fragments isolated from pre-immune serum were used as controls. Absorbance at 450 nm (OD 450 nm) indicates the level of immune response determined by ELISA.
  • FIG. 3 shows a schematic diagram of the chimeric antigen receptor (CAR) .
  • the vector construct includes sequences encoding: a GPC3-binding single-domain antibody (GPC3-sdAb) , a CD8 hinge, a CD8 transmembrane region (CD8 TM) , a 4-1BB intracellular co-stimulatory domain, and a CD3 ⁇ intracellular signaling sequence.
  • GPC3-sdAb GPC3-binding single-domain antibody
  • CD8 TM CD8 transmembrane region
  • 4-1BB intracellular co-stimulatory domain
  • CD3 ⁇ intracellular signaling sequence CD3 ⁇ intracellular signaling sequence
  • FIG. 4A shows a schematic diagram of an exemplary regular CAR and the corresponding vector construct.
  • the vector construct includes sequences encoding: a GPC3-binding domain, a CD8 or CD28 hinge, a CD8 or CD28 transmembrane region, a CD28 and/or 4-1BB intracellular co-stimulatory domain, and a CD3 ⁇ intracellular signaling sequence.
  • FIG. 4B shows a schematic diagram of an exemplary tandem CAR and the corresponding vector construct.
  • the vector construct includes sequences encoding: a first GPC3-binding domain (GPC3-binding domain 1) , a peptide linker, a second GPC3-binding domain (GPC3-binding domain 2) , a CD8 or CD28 hinge, a CD8 or CD28 transmembrane region, a CD28 and/or 4-1BB intracellular co-stimulatory domain, and a CD3 ⁇ intracellular signaling sequence.
  • FIG. 4C shows a schematic diagram of a dual CAR system and the corresponding vector construct.
  • the vector construct includes sequences encoding: (a) a first chimeric receptor comprising a first GPC3-binding domain (GPC3-binding domain 1) , a CD8 or CD28 hinge, a CD8 or CD28 transmembrane region, a first intracellular co-stimulatory domain (co-stimulatory domain 1) , a CD3 ⁇ intracellular signaling sequence; and (b) a second chimeric receptor comprising a second GPC3-binding domain (GPC3-binding domain 2) , a CD8 or CD28 hinge, a CD8 or CD28 transmembrane region, a second intracellular co-stimulatory domain (co-stimulatory domain 2) , a CD3 ⁇ intracellular signaling sequence.
  • P2A encodes a 2A self-cleaving peptide.
  • FIG. 5A shows the percentage of viable anti-GPC3 CAR-T cells with an extracellular sdAb on day 0 and day 5 after transduction.
  • Un-transduced T cells (UNT) and BMCAR T cells (BMCAR) were used as control cells.
  • FIG. 5B shows the expansion fold of anti-GPC3 CAR-T cells with an extracellular sdAb on day 0 and day 5 after transduction.
  • Un-transduced T cells (UNT) and BMCAR T cells (BMCAR) were used as control cells.
  • FIG. 6A shows representative in vitro cytotoxicity assay results showing cytolytic activity of CAR-T cells derived from various sdAbs against GPC3 + HCC cell line Huh7. Un-transduced T cells (UNT) were used as the negative control, and BMCAR T cells (BMCAR) were used as positive control.
  • UNT Un-transduced T cells
  • BMCAR BMCAR T cells
  • FIG. 6B shows representative in vitro cytotoxicity assay results showing cytolytic activity of CAR-T cells derived from various sdAbs against GPC3 + HCC cell line PLC-PRF5. Un-transduced T cells (UNT) were used as the negative control, and BMCAR T cells (BMCAR) were used as positive control.
  • UNT Un-transduced T cells
  • BMCAR BMCAR T cells
  • FIG. 6C shows representative in vitro cytotoxicity assay results showing cytolytic activity of CAR-T cells derived from various sdAbs against GPC3 - HCC cell line: SK-HEP-1. Un-transduced T cells (UNT) were used as the negative control, and BMCAR T cells (BMCAR) were used as positive control.
  • UNT Un-transduced T cells
  • BMCAR BMCAR T cells
  • FIG. 7A shows calculated cytotoxicity (Cytotoxicity%) of anti-GPC3 CAR-T cells in long-term co-culture assays with Huh7 cells.
  • the T cells were re-challenged for a total of 6 times.
  • Un-transduced T cells (UNT) were used as the negative control, and BMCAR T cells (BMCAR) were used as positive control.
  • FIG. 7B shows the expansion fold curves of anti-GPC3 CAR-T cells in long-term co-culture assays with Huh7 cells.
  • the T cells were re-challenged for a total of 6 times.
  • the curves reflected the calculated T cell proliferation.
  • FIG. 8A shows levels of IFN- ⁇ secreted by co-culturing anti-GPC3 CAR-T cells with Huh7 cells.
  • the first three rounds of supernatants from an in vitro cytotoxicity assay were assessed by an HTRF assay for IFN- ⁇ .
  • Supernatants from un-transduced T cells (UNT) were used as the negative control, and supernatants from BMCAR T cells (BMCAR) were used as positive control.
  • FIG. 8B shows levels of GM-CSF secreted by co-culturing anti-GPC3 CAR-T cells with Huh7 cells.
  • the first three rounds of supernatants from an in vitro cytotoxicity assay were assessed by an HTRF assay for GM-CSF.
  • Supernatants from un-transduced T cells (UNT) were used as the negative control, and supernatants from BMCAR T cells (BMCAR) were used as positive control.
  • FIG. 8C shows levels of TNF- ⁇ secreted by co-culturing anti-GPC3 CAR-T cells with Huh7 cells.
  • the first three rounds of supernatants from an in vitro cytotoxicity assay were assessed by an HTRF assay for TNF- ⁇ .
  • Supernatants from un-transduced T cells (UNT) were used as the negative control, and supernatants from BMCAR T cells (BMCAR) were used as positive control.
  • FIG. 9A shows calculated cytotoxicity (Cytotoxicity%) of tandem anti-GPC3 CAR-T cells with different extracellular sdAbs (Tan1, Tan2, Tan3, or Tan4) in long-term co-culture assays with Huh7 cells, as determined by FACS analysis.
  • the T cells were re-challenged for a total of 6 times. Un-transduced T cells (UNT) were used as the negative control, and BMCAR T cells (BMCAR) were used as positive control.
  • FIG. 9B shows the expansion fold curves of tandem anti-GPC3 CAR-T cells with different extracellular sdAbs (Tan1, Tan2, Tan3, or Tan4) in long-term co-culture assays with Huh7 cells, as determined by FACS analysis.
  • the T cells were re-challenged for a total of 6 times.
  • the curves reflected the calculated T cell proliferation.
  • FIG. 9C shows calculated cytotoxicity (Cytotoxicity%) of tandem anti-GPC3 CAR-T cells with different extracellular sdAbs (Tan5, Tan6, Tan7, or Tan8) in long-term co-culture assays with Huh7 cells, as determined by FACS analysis.
  • the T cells were re-challenged for a total of 6 times. Un-transduced T cells (UNT) were used as the negative control, and BMCAR T cells (BMCAR) were used as positive control.
  • FIG. 9D shows the expansion fold curves of tandem anti-GPC3 CAR-T cells with different extracellular sdAbs (Tan5, Tan6, Tan7, or Tan8) in long-term co-culture assays with Huh7 cells, as determined by FACS analysis.
  • the T cells were re-challenged for a total of 6 times.
  • the curves reflected the calculated T cell proliferation.
  • FIG. 10A shows calculated cytotoxicity (Cytotoxicity%) of regular anti-GPC3 CAR-T cells with a single extracellular sdAb (AS174556, AS162571, AS173786, or AS178894) or CD3 + tandem anti-GPC3 CAR-T cells with different extracellular sdAbs (Tan2, Tan6, or Tan8) in long-term co-culture assays with Huh7 cells, as determined by FACS analysis. Un-transduced T cells (UNT) were used as the negative control.
  • FIG. 10B shows the expansion fold curves of regular anti-GPC3 CAR-T cells with a single extracellular sdAb (AS174556, AS162571, AS173786, or AS178894) or tandem anti-GPC3 CAR-T cells with different extracellular sdAbs (Tan2, Tan6, or Tan8) in long-term co-culture assays with Huh7 cells, as determined by FACS analysis.
  • the T cells were re-challenged for a total of 6 times.
  • the curves reflected the calculated T cell proliferation.
  • FIG. 11A shows levels of IFN- ⁇ secreted by regular anti-GPC3 CAR-T cells with a single extracellular sdAb (AS174556, AS162571, AS173786, or AS178894) or tandem anti-GPC3 CAR-T cells with different extracellular sdAbs (Tan2, Tan6, or Tan8) in long-term co-culture assays with Huh7 cells.
  • R1, R2, R3, R4, R5, and R6 represent the 6 re-challenges.
  • Un-transduced T cells (UNT) were used as the negative control.
  • FIG. 11B shows levels of GM-CSF secreted by regular anti-GPC3 CAR-T cells with a single extracellular sdAb (AS174556, AS162571, AS173786, or AS178894) or tandem anti-GPC3 CAR-T cells with different extracellular sdAbs (Tan2, Tan6, or Tan8) in long-term co-culture assays with Huh7 cells.
  • R1, R2, R3, R4, R5, and R6 represent the 6 re-challenges.
  • Un-transduced T cells (UNT) were used as the negative control.
  • FIG. 11C shows levels of TNF- ⁇ secreted by regular anti-GPC3 CAR-T cells with a single extracellular sdAb (AS174556, AS162571, AS173786, or AS178894) or tandem anti-GPC3 CAR-T cells with different extracellular sdAbs (Tan2, Tan6, or Tan8) in long-term co-culture assays with Huh7 cells.
  • R1, R2, R3, R4, R5, and R6 represent the 6 re-challenges.
  • Un-transduced T cells (UNT) were used as the negative control.
  • FIG. 12 shows sequences of complementarity-determining region (CDR) and framework (FR) region sequences of various single-domain antibodies described herein.
  • FIG. 13 shows anti-tumor effect of tandem CAR T cells in huh7 xenograft model.
  • Un-transduced T cells (UNT) were used as the negative control, and BMCAR T cells (BMCAR) were used as positive control.
  • FIG. 14 lists amino acid sequences of sdAbs. CDR sequences are underlined.
  • FIG. 15 lists nucleic acid sequences encoding sdAbs.
  • FIG. 16 lists amino acid sequences of CARs with different extra-cellular sdAb.
  • FIG. 17 lists amino acid sequences of tandem CARs and BMCAR.
  • FIG. 18 lists sequences discussed in the disclosure.
  • HcAbs unique functional heavy (H) -chain only antibodies
  • the H chain of these homodimeric antibodies has one single antigen-binding domain, referred to as V H Hs, nanobodies or single-domain antibodies (sdAbs) .
  • V H Hs single antigen-binding domain
  • sdAbs single-domain antibodies
  • HcAbs do not interact with the light (L) chains owing to the deletion of the first constant domain and a reshaped surface at the V H H side.
  • single-domain antibodies targeting GPC3, and chimeric antigen receptors e.g., monovalent CAR, and multivalent CAR including bi-epitope CAR
  • chimeric antigen receptors e.g., monovalent CAR, and multivalent CAR including bi-epitope CAR
  • anti-GPC3 V H Hs anti-GPC3 V H Hs.
  • phage display library construction, CAR synthesis, in vitro and in vivo screening these antibodies and/or CARs were found to have good tumor killing activity, which can be used for treating cancer (e.g., HCC) .
  • cancer e.g., HCC
  • Glypican 3 (GPC3) , also called OCI-5, DGSX, GTR2-2, MXR7, SDYS, SGB, SGBS, and SGBS1, is a glycosylphosphatidylinositol-anchored cell surface protein consisting of a core protein and two heparan sulfate (HS) chains.
  • GPC3 belongs to the glypican-related integral membrane proteoglycan family, which includes six members (GPC1-GPC6) .
  • GPC3 is encoded at chromosome Xp26 adjacent to GPC4, and spans more than 500 kilobases.
  • isoform 2 GenBank Accession No.: NP_004475
  • isoform 2 which encodes a 70-kDa precursor core protein with 580 amino acids, is the most commonly expressed.
  • GPC3 is an oncofetal protein expressed in over 70%of HCC and other solid tumors including hepatoblastoma and lung squamous cell carcinoma. Its expression is not detected in nonmalignant adult tissues including normal liver. Mechanistically, GPC3 can promote tumor growth by modulating the Wnt/Frizzled signaling complex on HCC cells. Considering the highly specific expression of GPC3 in hepatocellular carcinoma, melanoma and other tumors, it is considered as a candidate target antigen for CAR-T cells tumor immunotherapy.
  • GPC3 Details of GPC3 and its functions can be found, e.g., in Filmus, Jorge, et al., Biochemical Journal 311.2 (1995) : 561-565; Capurro M. et al., Gastroenterology. 2003 Jul; 125 (1) : 89-97; Baumhoer D. et al., Am J Clin Pathol. 2008 Jun; 129 (6) : 899-906; Aviel-Ronen S. et al., Mod Pathol. 2008 Jul; 21 (7) : 817-25; Montalbano M. et al., Oncol Rep. 2017 Mar; 37 (3) : 1291-1300; Chen, C. et al., Clinical and Translational Medicine 6.1 (2017) : 1-6; and Li N. et al., Hepatology. 2019 Oct; 70 (4) : 1231-1245; each of which is incorporated herein by reference in its entirety.
  • GPC3-associated diseases include, but not limited to, cancer (e.g., liver cancer (e.g., hepatocellular carcinoma) , glioma, lung cancer (e.g., squamous cell lung cancer) , colorectal cancer, head and neck cancer, stomach cancer, esophageal cancer, renal cancer, urothelial cancer, testis cancer, breast cancer, pancreatic cancer, cervical cancer, endometrial cancer, ovarian cancer, thyroid cancer, gastrointestinal cancer, skin cancer, sarcoma, or urogenital cancer) , and Simpson–Golabi–Behmel syndrome.
  • cancer e.g., liver cancer (e.g., hepatocellular carcinoma)
  • lung cancer e.g., squamous cell lung cancer
  • colorectal cancer head and neck cancer
  • stomach cancer e.g., esophageal cancer
  • renal cancer e.g., squamous cell lung cancer
  • urothelial cancer
  • Monoclonal and recombinant antibodies are important tools in medicine and biotechnology. Like all mammals, camelids (e.g., llamas) can produce conventional antibodies made of two heavy chains and two light chains bound together with disulfide bonds in a Y shape (e.g., IgG1) . However, they also produce two unique subclasses of IgG: IgG2 and IgG3, also known as heavy chain IgG. These antibodies are made of only two heavy chains, which lack the CH1 region but still bear an antigen-binding domain at their N-terminus called V H H (or nanobody) . Conventional immunoglobulin (Ig) requires the association of variable regions from both heavy and light chains to allow a high diversity of antigen-antibody interactions.
  • heavy chain IgG The unique feature of heavy chain IgG is the capacity of their monomeric antigen binding regions to bind antigens with specificity, affinity, and especially diversity that are comparable to conventional antibodies without the need of pairing with another region. This feature is mainly due to a couple of major variations within the amino acid sequence of the variable region of the two heavy chains, which induce deep conformational changes when compared to conventional Ig. Major substitutions in the variable regions prevent the light chains from binding to the heavy chains, but also prevent unbound heavy chains from being recycled by the Immunoglobulin Binding Protein.
  • variable domain of these antibodies (designated V H H, sdAb, or nanobody) is the smallest antigen-binding domain generated by adaptive immune systems.
  • the third Complementarity Determining Region (CDR3) of the variable region of these antibodies has been found to be typically twice as long as the conventional ones. This results in an increased interaction surface with the antigen as well as an increased diversity of antigen-antibody interactions, which compensates the absence of the light chains.
  • CDR3 complementarity-determining region 3
  • V H Hs can extend into crevices on proteins that are not accessible to conventional antibodies, including functionally interesting sites such as the active site of an enzyme or the receptor-binding canyon on a virus surface.
  • an additional cysteine residue allow the structure to be more stable, thus increasing the strength of the interaction.
  • V H Hs offer numerous other advantages compared to conventional antibodies carrying variable domains (VH and VL) , including higher stability, solubility, expression yields, and refolding capacity, as well as better in vivo tissue penetration. Moreover, in contrast to the VH domains of conventional antibodies, V H Hs do not display an intrinsic tendency to bind to light chains. This facilitates the induction of heavy chain antibodies in the presence of a functional light chain loci. Further, since V H Hs do not bind to VL domains, it is much easier to reformat V H Hs into bispecific antibody constructs than constructs containing conventional VH-VL pairs or single domains based on VH domains.
  • the disclosure provides e.g., anti-GPC3 antibodies, the modified antibodies thereof, the chimeric antibodies thereof, and the humanized antibodies thereof.
  • the CDR sequences for AS162571, and AS162571 derived antibodies include CDRs of the V H H domain as set forth in SEQ ID NOs: 2, 4, and 6, respectively.
  • the CDR sequences for AS153931, and AS153931 derived antibodies include CDRs of the V H H domain as set forth in SEQ ID NOs: 9, 11, and 13, respectively.
  • the CDR sequences for AS153447, and AS153447 derived antibodies include CDRs of the V H H domain as set forth in SEQ ID NOs: 16, 18, and 20, respectively.
  • the CDR sequences for AS173786, and AS173786 derived antibodies include CDRs of the V H H domain as set forth in SEQ ID NOs: 23, 25, and 27, respectively.
  • the CDR sequences for AS185108, and AS185108 derived antibodies include CDRs of the V H H domain as set forth in SEQ ID NOs: 30, 32, and 34, respectively.
  • the CDR sequences for AS185124, and AS185124 derived antibodies include CDRs of the V H H domain as set forth in SEQ ID NOs: 37, 39, and 41, respectively.
  • the CDR sequences for AS174556, and AS174556 derived antibodies include CDRs of the V H H domain as set forth in SEQ ID NOs: 44, 46, and 48, respectively.
  • the CDR sequences for AS176653, and AS176653 derived antibodies include CDRs of the V H H domain as set forth in SEQ ID NOs: 51, 53, and 55, respectively.
  • the CDR sequences for AS178894, and AS178894 derived antibodies include CDRs of the V H H domain as set forth in SEQ ID NOs: 58, 60, and 62, respectively.
  • nucleic acid sequence and the encoded amino acid sequence for the V H H domain of AS162571 antibody is set forth in SEQ ID NO: 73 and SEQ ID NO: 64, respectively.
  • nucleic acid sequence and the encoded amino acid sequence for the V H H domain of AS153931 antibody is set forth in SEQ ID NO: 74 and SEQ ID NO: 65, respectively.
  • nucleic acid sequence and the encoded amino acid sequence for the V H H domain of AS153447 antibody is set forth in SEQ ID NO: 75 and SEQ ID NO: 66, respectively.
  • nucleic acid sequence and the encoded amino acid sequence for the V H H domain of AS173786 antibody is set forth in SEQ ID NO: 76 and SEQ ID NO: 67, respectively.
  • nucleic acid sequence and the encoded amino acid sequence for the V H H domain of AS185108 antibody is set forth in SEQ ID NO: 77 and SEQ ID NO: 68, respectively.
  • nucleic acid sequence and the encoded amino acid sequence for the V H H domain of AS185124 antibody is set forth in SEQ ID NO: 78 and SEQ ID NO: 69, respectively.
  • nucleic acid sequence and the encoded amino acid sequence for the V H H domain of AS174556 antibody is set forth in SEQ ID NO: 79 and SEQ ID NO: 70, respectively.
  • nucleic acid sequence and the encoded amino acid sequence for the V H H domain of AS176653 antibody is set forth in SEQ ID NO: 80 and SEQ ID NO: 71, respectively.
  • nucleic acid sequence and the encoded amino acid sequence for the V H H domain of AS178894 antibody is set forth in SEQ ID NO: 81 and SEQ ID NO: 72, respectively.
  • the amino acid sequences for various modified or humanized V H H are also provided.
  • the V H H of an antibody can have more than one version of humanized sequences.
  • the humanized V H H domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any sequence of SEQ ID NOS: 64-72.
  • the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three V H H domain CDRs selected from the group of SEQ ID NOs: 2, 4, and 6; SEQ ID NOs: 9, 11, and 13; SEQ ID NOs: 16, 18, and 20; SEQ ID NOs: 23, 25, and 27; SEQ ID NOs: 30, 32, and 34; SEQ ID NOs: 37, 39, and 41; SEQ ID NOs: 44, 46, and 48; SEQ ID NOs: 51, 53, and 55; and SEQ ID NOs: 58, 60, and 62.
  • these CDRs are determined by Kabat definition.
  • the antibodies can have a heavy chain single variable domain (V H H) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected V H H CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected V H H CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected V H H CDR3 amino acid sequence.
  • the selected V H H CDRs 1, 2, 3 amino acid sequences as determined by Kabat definition are shown in FIG. 12.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain single variable domain (V H H) containing one, two, or three of V H H CDR1 with zero, one or two amino acid insertions, deletions, or substitutions; V H H CDR2 with zero, one or two amino acid insertions, deletions, or substitutions; V H H CDR3 with zero, one or two amino acid insertions, deletions, or substitutions, wherein V H H CDR1, V H H CDR2, and V H H CDR3 are selected from the CDRs in FIG. 12.
  • V H H H heavy chain single variable domain
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain single variable domain (V H H) containing one, two, or three of the CDRs of SEQ ID NO: 2 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 4 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with zero, one or two amino acid insertions, deletions, or substitutions.
  • V H H heavy chain single variable domain
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain single variable domain (V H H) containing one, two, or three of the CDRs of SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 13 with zero, one or two amino acid insertions, deletions, or substitutions.
  • V H H heavy chain single variable domain
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain single variable domain (V H H) containing one, two, or three of the CDRs of SEQ ID NO: 16 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 18 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with zero, one or two amino acid insertions, deletions, or substitutions.
  • V H H heavy chain single variable domain
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain single variable domain (V H H) containing one, two, or three of the CDRs of SEQ ID NO: 23 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 25 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 27 with zero, one or two amino acid insertions, deletions, or substitutions.
  • V H H heavy chain single variable domain
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain single variable domain (V H H) containing one, two, or three of the CDRs of SEQ ID NO: 30 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 32 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 34 with zero, one or two amino acid insertions, deletions, or substitutions.
  • V H H heavy chain single variable domain
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain single variable domain (V H H) containing one, two, or three of the CDRs of SEQ ID NO: 37 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 39 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 41 with zero, one or two amino acid insertions, deletions, or substitutions.
  • V H H heavy chain single variable domain
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain single variable domain (V H H) containing one, two, or three of the CDRs of SEQ ID NO: 44 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 46 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 48 with zero, one or two amino acid insertions, deletions, or substitutions.
  • V H H heavy chain single variable domain
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain single variable domain (V H H) containing one, two, or three of the CDRs of SEQ ID NO: 51 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 53 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 55 with zero, one or two amino acid insertions, deletions, or substitutions.
  • V H H heavy chain single variable domain
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain single variable domain (V H H) containing one, two, or three of the CDRs of SEQ ID NO: 58 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 60 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 62 with zero, one or two amino acid insertions, deletions, or substitutions.
  • V H H heavy chain single variable domain
  • the insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.
  • the CDR is determined based on Kabat numbering scheme. In some embodiments, the CDR is determined based on Chothia numbering scheme. In some embodiments, the CDR is determined based on a combination numbering scheme.
  • the disclosure also provides antibodies or antigen-binding fragments thereof that bind to GPC3 (e.g., human GPC3) .
  • the antibodies or antigen-binding fragments thereof contain a heavy chain single variable region (V H H) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected V H H sequence.
  • the selected V H H sequence is SEQ ID NO: 64.
  • the selected V H H sequence is SEQ ID NO: 65.
  • the selected V H H sequence is SEQ ID NO: 66.
  • the selected V H H sequence is SEQ ID NO: 67.
  • the selected V H H sequence is SEQ ID NO: 68.
  • the selected V H H sequence is SEQ ID NO: 69. In some embodiments, the selected V H H sequence is SEQ ID NO: 70. In some embodiments, the selected V H H sequence is SEQ ID NO: 71. In some embodiments, the selected V H H sequence is SEQ ID NO: 72.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) .
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished, e.g., using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the disclosure also provides nucleic acid sequences encoding the immunoglobulin heavy chain single variable domains (V H Hs) described herein.
  • the V H Hs comprise CDRs as shown in FIG. 12, or has sequences as shown in FIG. 14.
  • the nucleic acid sequences encoding the V H Hs are listed in FIG. 15.
  • the antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments.
  • Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific) , human antibodies, chimeric antibodies (e.g., human-mouse chimera) , single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies) , and antigen-binding fragments thereof.
  • the antibodies or antigen-binding fragments thereof comprises an Fc region that can be originated from various types (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) , or subclass.
  • Fc region is originated from an IgG antibody or antigen-binding fragment thereof.
  • IgG subclasses The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al., Frontiers in Immunology 5 (2014) ; Irani, et al., Molecular Immunology 67.2 (2015) : 171-182; Shakib, Farouk, ed., The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.
  • the present disclosure also provides an antibody or antigen-binding fragment thereof that cross-competes with any antibody or antigen-binding fragment as described herein.
  • the cross-competing assay is known in the art, and is described e.g., in Moore et al., Journal of Virology 70.3 (1996) : 1863-1872, which is incorporated herein reference in its entirety.
  • the present disclosure also provides an antibody or antigen-binding fragment thereof that binds to the same epitope or region as any antibody or antigen-binding fragment as described herein.
  • the epitope binning assay is known in the art, and is described e.g., in Estep et al. MAbs. Vol. 5. No. 2. Taylor & Francis, 2013, which is incorporated herein reference in its entirety.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (V H H) CDR1 selected from SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, and 58. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (V H H) CDR2 selected from SEQ ID NOs: 4, 11, 18, 25, 32, 39, 46, 53, and 60. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (V H H) CDR3 selected from SEQ ID NOs: 6, 13, 20, 27, 34, 41, 48, 55, and 62.
  • the disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to GPC3.
  • the antibodies and antigen-binding fragments described herein are capable of binding to GPC3.
  • the antigen binding domains of the CARs or fragments thereof described herein can be derived from these antibodies or antigen binding fragments thereof.
  • the disclosure provides e.g., llama anti-GPC3 antibodies (e.g., AS162571, AS153931, AS153447, AS173786, AS185108, AS185124, AS174556, AS176653, or AS178894 sdAb) , and the chimeric antibodies thereof, and the humanized antibodies thereof.
  • the CDR sequences for AS162571, AS153931, AS153447, AS173786, AS185108, AS185124, AS174556, AS176653, or AS178894 sdAbs are provided in FIG. 12.
  • the V H Hs contain three hypervariable regions (e.g.., CDRs 1, 2, and 3) sandwiched between four conserved framework regions (e.g., FR1, FR2, FR3, and FR4) . Sequences of the CDRs 1, 2, 3 and FRs 1, 2, 3, 4 of the V H Hs described herein can be found in FIG. 12. These hypervariable regions, known as the complementary determining regions (CDRs) , form loops that comprise the antigen binding surface of the antibody.
  • the four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting the beta-sheet structure, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding region.
  • the CDRs are important for recognizing an epitope of an antigen.
  • an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen binding domain of an antibody.
  • the minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s secondary and tertiary structure.
  • the antibody or antigen-binding fragment thereof comprises an Fc region, e.g., as a llama immunoglobulin. In some embodiments, the antibody or antigen-binding fragment thereof does not comprise an Fc region, e.g., as a single-domain antibody. In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.
  • the antibody or antigen-binding fragment thereof described herein is a multi-specific antibody.
  • the multi-specific antibody is a bi-specific antibody.
  • Bi-specific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
  • the interface can contain at least a part of the CH3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan) .
  • Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) .
  • This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.
  • the Fc region is derived from human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibodies or antigen binding fragments do not have a functional Fc region. In some embodiments, the Fc region has LALA mutations (L234A and L235A mutations in EU numbering) , or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering) .
  • the antibodies or antigen binding fragments can specifically inhibit tumor growth (e.g., hepatocellular carcinoma growth) and enhance APC (e.g., DC cell) function, for example, inducing surface expression of costimulatory and MHC molecules, inducing production of proinflammatory cytokines, and/or enhancing T cell triggering function.
  • tumor growth e.g., hepatocellular carcinoma growth
  • enhance APC e.g., DC cell
  • the antigen binding fragment can form a part of a chimeric antigen receptor (CAR) .
  • the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 4-1BB, ICOS) .
  • the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-4-1BB or CD3z-CD28-OX40, to increase potency.
  • the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors as described herein.
  • Chimeric antigen receptors combine many facets of normal T cell activation into a single protein. They link an extracellular antigen recognition domain to an intracellular signaling domain, which activates the T cell when an antigen is bound. CARs typically have the following regions: an antigen binding domain, an extracellular hinge region, a transmembrane region, and an intracellular region. Intracellular region also known as intracellular signaling domain or intracellular signaling region or intracellular domain.
  • the antigen binding domain is exposed to the outside of the cell, in the ectodomain portion of the receptor. It interacts with potential target molecules and is responsible for targeting the CAR-T cell to any cell expressing a matching molecule.
  • the antigen binding domain is typically derived from the variable regions of a monoclonal antibody linked together as a single-chain variable fragment (scFv) .
  • An scFv is a chimeric protein made up of the light (VL) and heavy (VH) chains of immunoglobulins, connected with a short linker peptide.
  • the antigen binding domain comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) heavy chain single variable domains (V H Hs) .
  • the V H Hs are connected with a linker peptide (e.g., a flexible linker) .
  • the linker peptide between the two V H Hs includes hydrophilic residues with stretches of glycine and serine in it for flexibility as well as stretches of glutamate and lysine for added solubility.
  • the linker peptide comprises at least or about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 amino acid residues. In some embodiments, the linker peptide comprises at least or about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 30, or 40 glycine residues. In some embodiments, the linker peptide comprises at least or about 1, 2, 3, 4, 5, 6, 7, or 8 serine residues. In some embodiments, the linker peptide comprises or consists of both glycine and serine residues.
  • the linker peptide comprises or consists of a sequence that is at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, or 100%identical to GGGGS (SEQ ID NO: 99) or GGGGSGGGGSGGGGS (SEQ ID NO: 100) .
  • the linker sequence comprises at least 1, 2, 3, 4, 5, 6, 7, or 8 repeats of GGGGS (SEQ ID NO: 99) .
  • the linker sequence has no more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 amino acid residues.
  • the linker peptide comprises 1, 2, 3, 4, or 5 amino acid insertions, deletions, or substitutions.
  • the antigen binding domain specifically binds to GPC3 (e.g., human GPC3, or monkey (Rhesus macaque) GPC3) .
  • GPC3 e.g., human GPC3, or monkey (Rhesus macaque) GPC3 .
  • the hinge also called a spacer, is a small structural domain that sits between the antigen binding domain and the cell's outer membrane.
  • An ideal hinge enhances the flexibility of the antigen binding domain, reducing the spatial constraints between the CAR and its target antigen. This promotes antigen binding and synapse formation between the CAR-T cells and target cells.
  • Hinge sequences are often based on IgG hinge regions, or membrane-proximal regions from immune molecules including e.g., CD8 and CD28.
  • the transmembrane region is a structural component, consisting of a hydrophobic alpha helix that spans the cell membrane. It anchors the CAR to the plasma membrane, bridging the extracellular hinge and antigen binding domains with the intracellular signaling domain. This domain is essential for the stability of the receptor as a whole. Generally, the transmembrane domain from the most membrane-proximal component of the endodomain is used, but different transmembrane domains result in different receptor stability.
  • the CD28 transmembrane domain is known to result in a highly expressed, stable receptor.
  • the intracellular T cell signaling region lies in the receptor's endodomain, inside the cell. After an antigen is bound to the external antigen binding domain, CAR receptors cluster together and transmit an activation signal. Then the internal cytoplasmic end of the receptor perpetuates signaling inside the T cell.
  • Normal T cell activation relies on the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) present in the cytoplasmic domain of CD3-zeta. To mimic this process, CD3-zeta's cytoplasmic domain is commonly used as the main CAR endodomain component. T cells also require co-stimulatory molecules in addition to CD3 signaling in order to persist after activation.
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • the endodomain of CAR receptors typically also include one or more chimeric domains from co-stimulatory proteins.
  • Signaling domains from a wide variety of co-stimulatory molecules have been successfully tested, including CD28, CD27, CD134 (OX40) , ICOS, hematopoietic cell signal transducer (DAP10) and/or CD137 (4 ⁇ 1BB) .
  • the CAR molecules specifically binds to GPC3 (e.g., human GPC3) .
  • the CAR comprises the amino acid sequence set forth in any of SEQ ID NOs: 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98; or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity thereto.
  • antigen receptors including the hinge, the transmembrane domain, and the intracellular T cell signaling domain, and methods for engineering and introducing such receptors into cells, are described, for example, in Chandran et al., Immunological Reviews 290.1 (2019) : 127-147; Cartellieri, Marc, et al., BioMed Research International 2010 (2010) ; and PCT Publication No. WO2017173256A1; US2002/131960, US2013/287748, US2013/0149337, U.S. 6,451,995, U.S. 7,446,190, and U.S. 8,252,592; each of which is incorporated herein by reference in its entirety.
  • the disclosure provides chimeric antigen receptors (CARs) or fragments thereof that specifically bind to GPC3.
  • CARs chimeric antigen receptors
  • the CARs or fragments thereof described herein are capable of binding to GPC3.
  • the disclosure provides CARs or fragments thereof, comprising (a) an extracellular antigen-binding domain that specifically recognizes Glypican 3 (GPC3) ; (b) a transmembrane region; and/or (c) an intracellular signaling domain.
  • the antigen-binding domain includes a heavy chain single variable domain (V H H) .
  • the antigen-binding domain of the CARs or fragments thereof described herein are identical to any of the V H Hs described herein (e.g., AS162571, AS153931, AS153447, AS173786, AS185108, AS185124, AS174556, AS176653, or AS178894) , or a humanized antibody thereof.
  • the antigen-binding domain of the CARs or fragments thereof described herein comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) V H Hs that are connected with the linker peptide described herein.
  • the CDR sequences of the antigen-binding domain e.g., a V H H for AS162571 CAR, or related antigen binding fragment thereof include V H H CDR1, V H H CDR2, and V H H CDR3 comprising or consisting of SEQ ID NOs: 2, 4, and 6, respectively.
  • the CDR sequences of the antigen-binding domain e.g., a V H H for AS153931 CAR, or related antigen binding fragment thereof include V H H CDR1, V H H CDR2, and V H H CDR3 comprising or consisting of SEQ ID NOs: 9, 11, and 13, respectively.
  • the CDR sequences of the antigen-binding domain e.g., a V H H for AS153447 CAR, or related antigen binding fragment thereof include V H H CDR1, V H H CDR2, and V H H CDR3 comprising or consisting of SEQ ID NOs: 16, 18, and 20, respectively.
  • the CDR sequences of the antigen-binding domain e.g., a V H H for AS173786 CAR, or related antigen binding fragment thereof include V H H CDR1, V H H CDR2, and V H H CDR3 comprising or consisting of SEQ ID NOs: 23, 25, and 27, respectively.
  • the CDR sequences of the antigen-binding domain e.g., a V H H for AS185108 CAR, or related antigen binding fragment thereof include V H H CDR1, V H H CDR2, and V H H CDR3 comprising or consisting of SEQ ID NOs: 30, 32, and 34, respectively.
  • the CDR sequences of the antigen-binding domain e.g., a V H H for AS185124 CAR, or related antigen binding fragment thereof include V H H CDR1, V H H CDR2, and V H H CDR3 comprising or consisting of SEQ ID NOs: 37, 39, and 41, respectively.
  • the CDR sequences of the antigen-binding domain e.g., a V H H for AS174556 CAR, or related antigen binding fragment thereof include V H H CDR1, V H H CDR2, and V H H CDR3 comprising or consisting of SEQ ID NOs: 44, 46, and 48, respectively.
  • the CDR sequences of the antigen-binding domain e.g., a V H H for AS176653 CAR, or related antigen binding fragment thereof include V H H CDR1, V H H CDR2, and V H H CDR3 comprising or consisting of SEQ ID NOs: 51, 53, and 55, respectively.
  • the CDR sequences of the antigen-binding domain e.g., a V H H for AS178894 CAR, or related antigen binding fragment thereof include V H H CDR1, V H H CDR2, and V H H CDR3 comprising or consisting of SEQ ID NOs: 58, 60, and 62, respectively.
  • amino acid sequence of V H H in the antigen-binding domain for AS162571 CAR, or related antigen binding fragment thereof is set forth in SEQ ID NO: 64.
  • amino acid sequence of V H H in the antigen-binding domain for AS153931 CAR, or related antigen binding fragment thereof is set forth in SEQ ID NO: 65.
  • V H H in the antigen-binding domain for AS153447 CAR, or related antigen binding fragment thereof is set forth in SEQ ID NO: 66.
  • V H H in the antigen-binding domain for AS173786 CAR, or related antigen binding fragment thereof is set forth in SEQ ID NO: 67.
  • V H H in the antigen-binding domain for AS185108 CAR, or related antigen binding fragment thereof is set forth in SEQ ID NO: 68.
  • V H H in the antigen-binding domain for AS185124 CAR, or related antigen binding fragment thereof is set forth in SEQ ID NO: 69.
  • amino acid sequence of V H H in the antigen-binding domain for AS174556 CAR, or related antigen binding fragment thereof is set forth in SEQ ID NO: 70.
  • V H H in the antigen-binding domain for AS176653 CAR, or related antigen binding fragment thereof is set forth in SEQ ID NO: 71.
  • amino acid sequence of V H H in the antigen-binding domain for AS178894 CAR, or related antigen binding fragment thereof is set forth in SEQ ID NO: 72.
  • the amino acid sequences for V H H of the antigen-binding domain for the CAR, or related antigen binding fragment thereof are humanized (e.g., a sequence can be modified with different amino acid substitutions) .
  • the V H H can have more than one version of humanized sequences.
  • the humanized V H H is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 64-72.
  • the CAR, related antibody or antigen binding fragment thereof described herein can also contain one, two, or three V H H domain CDRs selected from the group of SEQ ID NOs: 2, 4, and 6; SEQ ID NOs: 9, 11, and 13; SEQ ID NOs: 16, 18, and 20; SEQ ID NOs: 23, 25, and 27; SEQ ID NOs: 30, 32, and 34; SEQ ID NOs: 37, 39, and 41; SEQ ID NOs: 44, 46, and 48; SEQ ID NOs: 51, 53, and 55; and SEQ ID NOs: 58, 60, and 62.
  • the CAR, related antibody or antigen binding fragment thereof described herein can have a heavy chain single variable domain (V H H) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR3 amino acid sequence.
  • the selected V H H CDRs 1, 2, 3 amino acid sequences are shown in FIG. 12.
  • the CAR, related antibody or antigen binding fragment thereof described herein contains a V H H containing one, two, or three of the V H H CDR1 with zero, one or two amino acid insertions, deletions, or substitutions; V H H CDR2 with zero, one or two amino acid insertions, deletions, or substitutions; V H H CDR3 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the disclosure also provides CARs or fragments thereof that bind to GPC3.
  • the CAR, related antibody or antigen binding fragment thereof contains a heavy chain single variable region (V H H) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected V H H sequence.
  • V H H heavy chain single variable region
  • the selected V H H sequence is SEQ ID NO: 64.
  • the selected V H H sequence is SEQ ID NO: 65.
  • the selected V H H sequence is SEQ ID NO: 66.
  • the selected V H H sequence is SEQ ID NO: 67.
  • the selected V H H sequence is SEQ ID NO: 68.
  • the selected V H H sequence is SEQ ID NO: 69. In some embodiments, the selected V H H sequence is SEQ ID NO: 70. In some embodiments, the selected V H H sequence is SEQ ID NO: 71. In some embodiments, the selected V H H sequence is SEQ ID NO: 72.
  • the amino acid sequence for AS162571 CAR is set forth in SEQ ID NO: 82.
  • the amino acid sequence for AS153931 CAR is set forth in SEQ ID NO: 83.
  • the amino acid sequence for AS153447 CAR is set forth in SEQ ID NO: 84.
  • the amino acid sequence for AS173786 CAR is set forth in SEQ ID NO: 85.
  • the amino acid sequence for AS185108 CAR is set forth in SEQ ID NO: 86.
  • the amino acid sequence for AS185124 CAR is set forth in SEQ ID NO: 87.
  • the amino acid sequence for AS174556 CAR is set forth in SEQ ID NO: 88.
  • the amino acid sequence for AS176653 CAR is set forth in SEQ ID NO: 89.
  • the amino acid sequence for AS178894 CAR is set forth in SEQ ID NO: 90.
  • CARs comprising 2 V H Hs that are connected with the linker peptide described herein (i.e., tandem CARs) .
  • the amino acid sequence for Tan1 CAR is set forth in SEQ ID NO: 91; the amino acid sequence for Tan2 CAR is set forth in SEQ ID NO: 92; the amino acid sequence for Tan3 CAR is set forth in SEQ ID NO: 93; the amino acid sequence for Tan4 CAR is set forth in SEQ ID NO: 94; the amino acid sequence for Tan5 CAR is set forth in SEQ ID NO: 95; the amino acid sequence for Tan6 CAR is set forth in SEQ ID NO: 96; the amino acid sequence for Tan7 CAR is set forth in SEQ ID NO: 97; and the amino acid sequence for Tan8 CAR is set forth in SEQ ID NO: 98.
  • the extracellular antigen-binding domain of the tandem CAR described herein comprises two heavy chain single variable domains (V H Hs) .
  • the V H Hs are selected from the group consisting of AS162571, AS153931, AS153447, AS173786, AS185108, AS185124, AS174556, AS176653, and/or AS178894.
  • the first and the second V H H are listed in the table below.
  • CARs or fragments thereof comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98.
  • the CAR described herein comprises an amino acid sequence as set forth in SEQ ID NO: 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98; optionally with about or no more than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid insertions, deletions, or substitutions.
  • the chimeric antigen receptors (CARs) or fragments thereof described herein comprises a hinge region.
  • the hinge region is a membrane-proximal region from CD8, and/or CD28, or an IgG hinge region, or any combination thereof.
  • the hinge region is a membrane-proximal region of CD8 (e.g., human CD8) .
  • the hinge region is a fusion peptide comprising all or a portion of the membrane-proximal region of CD28 (e.g., human CD28) and all or a portion of the membrane-proximal region of CD8 (e.g., human CD8) .
  • the hinge region comprises the membrane-proximal regions of both CD8 and CD28.
  • the chimeric antigen receptors (CARs) or fragments thereof described herein comprises a transmembrane region.
  • the transmembrane domain is a transmembrane domain of 4-1BB/CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8, CD8 alpha, CD9, CD16, CD19, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or a zeta chain of a T cell receptor, or any combination thereof.
  • the transmembrane region is a transmembrane region from CD8 (e.g., human CD8) .
  • the hinge region and the transmembrane region are directly joined.
  • the joined hinge region and the transmembrane region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 101.
  • the transmembrane region is a fusion peptide comprising all or a portion of the transmembrane region of CD28 (e.g., human CD28) and all or a portion of the transmembrane region of CD8 (e.g., human CD8) .
  • the transmembrane region comprises the transmembrane regions of both CD8 and CD28.
  • the chimeric antigen receptors (CARs) or fragments thereof described herein comprises an intracellular signaling domain.
  • the intracellular signaling domain comprises an activating cytoplasmic signaling domain, which is capable of inducing a primary activation signal in an immune cell (e.g., a T cell) .
  • the activating cytoplasmic signaling domain is a T cell receptor (TCR) component.
  • the activating cytoplasmic signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM) .
  • ITAM immunoreceptor tyrosine-based activation motif
  • the intracellular signaling domain comprises an amino acid sequence derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (ICOS) , FceRI, CD66d, DAP10, DAP12, or combinations thereof.
  • the intracellular signaling domain comprises a functional signaling domain of CD3 zeta (e.g., a human CD3 zeta) .
  • the intracellular signaling domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 103.
  • the chimeric antigen receptors (CARs) or fragments thereof described herein comprises a costimulatory signaling domain.
  • the costimulatory signaling domain is between the transmembrane domain and the intracellular signaling domain.
  • the costimulatory signaling domain comprises a functional signaling domain from a protein selected from the group consisting of a MHC class I molecule, a TNF receptor protein, an Immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation molecule (SLAM protein) , an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1, CD11a/CD18, 4-1BB (CD137) , B7-H3, CDS, ICAM-1, ICOS (CD278) , GITR, BAFFR, LIGHT, HVEM (LIGHTR) , KIRDS2, SLAMF7, NKp80 (KLRF1) , NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2
  • the costimulatory signaling domain comprises a functional signaling domain from OX40, CD28, 4-1BB, ICOS, or a signaling portion thereof.
  • the costimulatory signaling domain comprises an intracellular signaling domain of 4-1BB (e.g., human 4-1BB) .
  • the costimulatory signaling domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 102.
  • the costimulatory signaling domain comprises an intracellular signaling domain of CD28 (e.g., human CD28) .
  • the costimulatory signaling domain comprises intracellular signaling domains of both CD28 (e.g., human CD28) and 4-1BB (e.g., human 4-1BB) .
  • the costimulatory signaling domain is a fusion peptide comprising all or a portion of the intracellular signaling domain of CD28 (e.g., human CD28) and all or a portion of the intracellular signaling domain of 4-1BB (e.g., human 4-1BB) .
  • the chimeric antigen receptors (CAR) described herein has a schematic structure shown in FIG. 3.
  • the CAR comprises, preferably from N-terminus to C-terminus, an anti-GPC3 sdAb, a hinge region, a transmembrane region, a co-stimulatory domain, and an activating cytoplasmic signaling domain.
  • the CAR further comprises a signal peptide and/or a tag.
  • the signal peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 104.
  • the anti-GPC3 sdAb is any one of the anti-GPC3 sdAb or V H H described herein.
  • both the hinge region and the transmembrane region are derived from CD8, and comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 101.
  • the co-stimulatory domain is derived from 4-1BB and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 102.
  • the activating cytoplasmic signaling domain is derived from CD3 zeta and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 103.
  • the tag is FLAG tag, the FLAG tag comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 105.
  • the chimeric antigen receptors (CAR) described herein has a schematic structure shown in FIG. 4A.
  • the CAR comprises, preferably from N-terminus to C-terminus, a GPC3-binding domain (e.g., an anti-GPC3 sdAb) , a hinge region, a transmembrane region, a co-stimulatory domain, and an activating cytoplasmic signaling domain.
  • the CAR further comprises a signal peptide and/or a tag.
  • the signal peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 104.
  • the GPC3-binding domain (e.g., an anti-GPC3 sdAb) is any one of the anti-GPC3 sdAb or V H H described herein.
  • the hinge region is derived from CD8 or CD28, and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 106 or 108.
  • the transmembrane region is derived from CD8 or CD28, and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 107 or 109.
  • the co-stimulatory domain is derived from 4-1BB and/or CD28 (e.g., a fusion peptide) , and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 102, 110 or 111.
  • the activating cytoplasmic signaling domain is derived from CD3 zeta and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 103.
  • the tag is FLAG tag, the FLAG tag comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 105.
  • the chimeric antigen receptors (CAR) described herein has a schematic structure shown in FIG. 4B.
  • the CAR comprises, preferably from N-terminus to C-terminus, a first GPC3-binding domain (e.g., a first V H H) , a linker peptide, a second GPC3-binding domain (e.g., a second V H H) , a hinge region, a transmembrane region, a co-stimulatory domain, and an activating cytoplasmic signaling domain.
  • the CAR further comprises a signal peptide and/or a tag.
  • the signal peptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 104.
  • the first and second GPC3-binding domains are selected from any of the V H H combinations shown in Table 1.
  • the linker peptide is any of linker peptide disclosed herein, e.g., an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 99 or 100.
  • the hinge region is derived from CD8 and/or CD28, and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 106 or 108.
  • the transmembrane region is derived from CD8 and/or CD28, and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 107 or 109.
  • the co-stimulatory domain is derived from 4-1BB and/or CD28 (e.g., a fusion peptide) , and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 102, 110, or 111.
  • the activating cytoplasmic signaling domain is derived from CD3 zeta and comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 103.
  • the tag is Flag tag, the Flag tag comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 105.
  • the engineered cells (e.g., CAR-T cells) described herein can express the same type of CAR. In some embodiments, the engineered cells (e.g., CAR-T cells) described herein can express two or more types of CARs. In some embodiments, the combination of different types of CARs with different antigen binding domains are listed in Table 1. In some embodiments, the expressed CARs form a dimer (e.g., a homodimer or a heterodimer) on the surface of the engineered cells described herein.
  • a dimer e.g., a homodimer or a heterodimer
  • the hinge region, transmembrane region, and/or intracellular signaling domain (e.g., costimulatory signaling domain and/or activating cytoplasmic signaling domain) of CARs or fragments thereof described herein are derived from a first generation, a second generation, a third generation, or a fourth generation CAR structure. Details of the structural features of CARs can be found, e.g., in Jackson, Hollie J., et al., Nature Reviews Clinical Oncology 13.6 (2016) : 370; and Subklewe, Marion, et al., Transfusion Medicine and Hemotherapy 46.1 (2019) : 15-24; each of which is incorporated herein by reference.
  • the CAR, antibodies, or antigen-binding fragments thereof as described herein can increase immune response, activity or number of immune cells (e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds, as compared to that of immune cells that do not express the CAR, antibodies, or antigen-binding fragments thereof.
  • immune cells e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen presenting cells
  • KD (Kd) for the antibody, antigen-binding fragments thereof, or molecules derived therefrom is less than 1 ⁇ 10 -6 M, less than 1 ⁇ 10 -7 M, less than 1 ⁇ 10 -8 M, less than 1 ⁇ 10 -9 M, or less than 1 ⁇ 10 -10 M.
  • the KD is less than 100 nM, 50nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM.
  • KD is greater than 1 ⁇ 10 -7 M, greater than 1 ⁇ 10 -8 M, greater than 1 ⁇ 10 -9 M, greater than 1 ⁇ 10 -10 M, greater than 1 ⁇ 10 -11 M, or greater than 1 ⁇ 10 -12 M.
  • General techniques for measuring the affinity of an antibody for an antigen include, e.g., enzyme-linked immunosorbent assay (ELISA) , Radioimmunoassay (RIA) , fluorescence-activated cell sorting (FACS) , and surface plasmon resonance (SPR) .
  • ELISA enzyme-linked immunosorbent assay
  • RIA Radioimmunoassay
  • FACS fluorescence-activated cell sorting
  • SPR surface plasmon resonance
  • the antibody binds to human GPC3.
  • the antibody binds to monkey GPC3 (e.g., Rhesus macaque GPC3) .
  • the antibody binds to a cell expressing GPC3 (e.g., HepG2 cell) .
  • engineered cells e.g., immune cells, T cells, NK cells, tumor-infiltrating lymphocytes
  • CAR CAR
  • various proteins as described herein.
  • engineered cells can be used to treat various disorders or disease as described herein (e.g., GPC3-associated cancer) .
  • the cell that is engineered can be obtained from e.g., humans and non-human animals.
  • the cell that is engineered can be obtained from bacteria, fungi, humans, rats, mice, rabbits, monkeys, pig or any other species.
  • the cell is from humans, rats or mice.
  • the cells are mouse lymphocytes and engineered (e.g., transduced) to express the CAR, or antigen-binding fragment thereof.
  • the cell is obtained from humans.
  • the cell that is engineered is a blood cell.
  • the cell is a leukocyte (e.g., a T cell) , lymphocyte or any other suitable blood cell type.
  • the cell is a peripheral blood cell. In some embodiments, the cell is a tumor-infiltrating lymphocyte (TIL) . In some embodiments, the cell is a T cell, B cell or NK cell. In some embodiments, the cells are human peripheral blood mononuclear cells (PBMCs) . In some embodiments, the human PBMCs are CD3+ cells. In some embodiments, the human PBMCs are CD8+ cells or CD4+ cells.
  • TIL tumor-infiltrating lymphocyte
  • the cell is a T cell, B cell or NK cell.
  • the cells are human peripheral blood mononuclear cells (PBMCs) . In some embodiments, the human PBMCs are CD3+ cells. In some embodiments, the human PBMCs are CD8+ cells or CD4+ cells.
  • the cell is a T cell.
  • the T cells can express a cell surface receptor that recognizes a specific antigenic moiety on the surface of a target cell.
  • the cell surface receptor can be a wild type or recombinant T cell receptor (TCR) , a chimeric antigen receptor (CAR) , or any other surface receptor capable of recognizing an antigenic moiety that is associated with the target cell.
  • T cells can be obtained by various methods known in the art, e.g., in vitro culture of T cells (e.g., tumor infiltrating lymphocytes) isolated from patients. Genetically modified T cells can be obtained by transducing T cells (e.g., isolated from the peripheral blood of patients) , with a viral vector.
  • the T cells are CD4+ T cells, CD8+ T cells, or regulatory T cells.
  • the T cells are T helper type 1 T cells and T helper type 2 T cells.
  • the T cell expressing this receptor is an ⁇ -T cell. In alternate embodiments, the T cell expressing this receptor is a ⁇ -T cell.
  • the T cells are central memory T cells. In some embodiments, the T cells are effector memory T cells. In some embodiments, the T cells are T cells.
  • the cell is an NK cell.
  • preparation of the engineered cells includes one or more culture and/or preparation steps.
  • the cells for introduction of the binding molecule, e.g., CAR can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
  • the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered.
  • the subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
  • the cells are stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs) .
  • the cells can be primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen.
  • the stem cells are cultured with additional differentiation factors to obtain desired cell types (e.g., T cells) .
  • the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers can be used. In some embodiments, the separation is affinity-or immunoaffinity-based separation.
  • the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
  • Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
  • the genetic engineering generally involves introduction of a nucleic acid encoding the therapeutic molecule, e.g., CAR, polypeptides, fusion proteins, into the cell, such as by retroviral transduction, transfection, or transformation.
  • gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical application.
  • recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40) , adenoviruses, adeno-associated virus (AAV) .
  • recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors.
  • the retroviral vector has a long terminal repeat sequence (LTR) , e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV) , myeloproliferative sarcoma virus (MPSV) , murine embryonic stem cell virus (MESV) , murine stem cell virus (MSCV) , or spleen focus forming virus (SFFV) .
  • LTR long terminal repeat sequence
  • MoMLV Moloney murine leukemia virus
  • MPSV myeloproliferative sarcoma virus
  • MSV murine embryonic stem cell virus
  • MSCV murine stem cell virus
  • SFFV spleen focus forming virus
  • retroviral vectors are derived from murine retroviruses.
  • the retroviruses include those derived from any avian or mammalian cell source.
  • the retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including
  • the vector is a lentivirus vector.
  • recombinant nucleic acids are transferred into T cells via electroporation.
  • recombinant nucleic acids are transferred into T cells via transposition.
  • Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection, protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment and strontium phosphate DNA co-precipitation. Many of these methods are descried e.g., in WO2019195486, which is incorporated herein by reference in its entirety.
  • the T cells are pre-activated, e.g., using anti-CD3/CD28 particles, for about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 60 hours prior to transduction.
  • the transduced T cells are harvested on day 5, day 6, day 7, day 8, day 9, day 10, day 11, or day 12 post transduction.
  • the transfection efficiency of the virus-infected T cells described herein is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%.
  • the viability of the transduced T cells is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%on day 0, day 1, day 2, day 3, day 4, or day 5 post transduction.
  • the viability of the transduced T cells is at least or about 80%, at least or about 90%, at least or about 100%, at least or about 110%, at least or about 120%as compared to the viability of untransduced T cells, on day 0, day 1, day 2, day 3, day 4, or day 5 (e.g., on day 5) post transduction.
  • the T cell expansion fold is at least 1 fold, 2 folds, 3 folds, 4 folds, 5 folds, 10 folds, 15 folds, 20 folds, 25 folds, 30 folds, 35 folds, 40 folds, 45 folds, or 50 folds, on day 0, day 1, day 2, day 3, day 4, or day 5 post transduction.
  • the T cell expansion fold of the transduced T cells is at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%as compared to that of untransduced T cells, on day 0, day 1, day 2, day 3, day 4, or day 5 (e.g., on day 5) post transduction.
  • populations of engineered cells, compositions containing such cells and/or enriched for such cells such as in which cells expressing the CAR make up at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more percent of the total cells in the composition or cells of a certain type such as T cells, CD8+ or CD4+ cells.
  • the engineered cells e.g., CAR-T cells
  • the engineered cells are co-cultured with target cells for at least or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 16 hours, 18 hours, 1 day, 2 days, 3 days, or longer, such that the engineered cells (e.g., CAR-T cells) can be activated.
  • the target cells express GPC3, e.g., Huh7 or PLC-PRF5. In some embodiments, the target cells do not express GPC3, e.g., SK-HEP-1.
  • the in vitro cytotoxicity of the engineered cells described herein is determined. In some embodiments, the engineered cells are incubated with the target cells at an E: T ratio of about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 0.9: 1, about 0.8: 1, about 0.7: 1, about 0.6: 1, about 0.5: 1, about 0.4: 1, about 0.3: 1, about 0.2: 1, or about 0.1: 1. In some embodiments, the incubation is about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 20 hours, about 22 hours, about 24 hours, about 36 hours, or about 48 hours.
  • the calculated percentage of cytotoxicity is at least 80%, at least 85%, or at least 90%when the engineered cells are incubated with Huh7 cells at an E: T ratio of 3: 1. In some embodiments, the calculated percentage of cytotoxicity (Cytotoxicity%) is at least 70%, at least 75%, at least 80%when the engineered cells are incubated with Huh7 cells at an E: T ratio of 1: 1. In some embodiments, the calculated percentage of cytotoxicity (Cytotoxicity%) is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%when the engineered cells are incubated with Huh7 cells at an E: T ratio of 0.3: 1.
  • the calculated percentage of cytotoxicity is at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%when the engineered cells are incubated with PLC-PRF5 cells at an E: T ratio of 3: 1. In some embodiments, the calculated percentage of cytotoxicity (Cytotoxicity%) is at least 30%, at least 35%, at least 40%when the engineered cells are incubated with PLC-PRF5 cells at an E: T ratio of 1: 1. In some embodiments, the calculated percentage of cytotoxicity (Cytotoxicity%) is at least 10%, at least 15%, at least 20%, or at least 25%, when the engineered cells are incubated with PLC-PRF5 cells at an E: T ratio of 0.3: 1.
  • the calculated percentage of cytotoxicity (Cytotoxicity%) of the engineered cells is at least 1 fold, 2 folds, 3 folds, 4 folds, 5 folds, 6 folds, 7 folds, 8 folds, 9 folds, or 10 folds as compared to that of the untransduced cells, when incubated with Huh7 or PLC-PRF5 cells at an E: T ratio of 3: 1, 1: 1, or 0.3: 1.
  • the calculated percentage of cytotoxicity is less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%or less than 1%when the engineered cells are incubated with target cells that do not express GPC3 (e.g., SK-HEP-1 cells) at an E: T ratio of 3: 1, 1: 1, or 0.3: 1.
  • GPC3 e.g., SK-HEP-1 cells
  • the long-term cytotoxicity of the engineered cells is determined, e.g., by re-challenging the engineered cells.
  • Exemplary re-challenging procedures of CAR-T cells can be found, e.g., in Wang, Dongrui, et al., Journal of Visualized Experiments: JoVE 144 (2019) ; Wang D, et al., JCI Insight 2018, 3 (10) ; Lange et al., Cancer Discov. 2021 Feb 9, candisc. 0896.2020; each of which is incorporated herein by reference in its entirety.
  • the engineered cells e.g., CAR-T cells
  • target cells e.g., Huh7 cells
  • the engineered cells are re-challenged by harvesting a portion of the cell pool and adding fresh target cells (e.g., Huh7 cells) at the same E: T ratio.
  • the engineered cells are re-challenged for at least 1, 2, 3, 4, 5, or 6 times.
  • the calculated cytotoxicity (Cytotoxicity%) is determined after each re-challenge.
  • the calculated cytotoxicity of the engineered cells described herein is at least 80%, at least 90%, or at least 95%.
  • the calculated cytotoxicity of the engineered cells described herein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.
  • the calculated cytotoxicity of the engineered cells described herein is at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments, after the fifth re-challenge, the calculated cytotoxicity of the engineered cells described herein is at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
  • the calculated cytotoxicity of the engineered cells described herein is at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.
  • the maximum re-challenge number (i.e., the number of re-challenge times before tumor cells outgrow) of the engineered cells described herein is at least 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times.
  • the calculated cytotoxicity of the engineered cells having a tandem CAR described herein is at least 1 fold, at least 2 folds, at least 3 folds, at least 4 folds, or at least 5 folds as compared to that of the engineered cells having a CAR with a single extracellular sdAb (e.g., any one of AS162571, AS153931, AS153447, AS173786, AS185108, AS185124, AS174556, AS176653, and AS178894 CARs) , after 1 re-challenge, 2 re-challenges, 3 re-challenges, 4 re-challenges, 5 re-challenges, or 6 re-challenges.
  • a single extracellular sdAb e.g., any one of AS162571, AS153931, AS153447, AS173786, AS185108, AS185124, AS174556, AS176653, and AS178894 CARs
  • the engineered cells e.g., CAR-T cells
  • target cells e.g., Huh7 cells
  • population of the engineered cells increases by at least or about 1 fold, 2 folds, 3 folds, 4 folds, 5 folds, 10 folds, 20 folds, 30 folds, 40 folds, 50 folds, 60 folds, 70 folds, 80 folds, 90 folds, 100 folds, 150 folds, 200 folds, or more, after 1 re-challenge, 2 re-challenges, 3 re-challenges, 4 re-challenges, 5 re-challenges, or 6 re-challenges, as compared to the initial population of the engineered cells.
  • the engineered cells e.g., CAR-T cells
  • target cells e.g., Huh7 cells
  • population of the calculated cytotoxicity of the engineered cells having a tandem CAR described herein is at least 1 fold, at least 2 folds, at least 3 folds, at least 4 folds, or at least 5 folds as compared to that of the engineered cell having a CAR with a single extracellular sdAb (e.g., any one of AS162571, AS153931, AS153447, AS173786, AS185108, AS185124, AS174556, AS176653, and AS178894 CARs)
  • a single extracellular sdAb e.g., any one of AS162571, AS153931, AS153447, AS173786, AS185108, AS185124, AS174556, AS176653, and AS178894 CARs
  • concentration of the cytokines (e.g., IFN- ⁇ , GM-CSF, and/or TNF- ⁇ ) released by the engineered cells (e.g., CAR-T cells) described herein is determined by homogeneous time resolved fluorescence (HTRF) assays.
  • HTRF time resolved fluorescence
  • the engineered cells e.g. CAR-T cells
  • increase cytokine e.g., IFN- ⁇ , GM-CSF, and/or TNF- ⁇
  • cytokine e.g., IFN- ⁇ , GM-CSF, and/or TNF- ⁇
  • the engineered cells increase cytokine (e.g., IFN- ⁇ , GM-CSF, and/or TNF- ⁇ ) expression or secretion by at least or about 1 fold, 2 folds, 3 folds, 4 folds, 5 folds, 10 folds, 20 folds, 30 folds, 40 folds, 50 folds, 60 folds, 70 folds, 80 folds, 90 folds, 100 folds, 500 folds, 1000 folds, 2000 folds, 3000 folds, 4000 folds, 5000 folds, 10000 folds, or more when co-cultured with the target cells (e.g., Huh7 or PLC-PRF-5) , as compared to the cytokine expression or secret
  • cytokine e.g., IFN- ⁇ , GM-CSF, and/or TNF- ⁇ expression or secretion of the engineered cells having a tandem CAR described herein (e.g., any one of Tan1, Tan2, Tan3, Tan4, Tan5, Tan6, Tan7, and Tan8 CARs) is at least or about 100%, at least or about 110%, at least or about 120%, at least or about 130%, at least or about 140%, at least or about 150%, or more, as compared to that of the engineered cell having a CAR with a single extracellular sdAb (e.g., any one of AS162571, AS153931, AS153447, AS173786, AS185108, AS185124, AS174556, AS176653, and AS178894 CARs) , after 1 re-challenge, 2 re-challenges, 3 re-challenges, 4 re-challenges, 5 re-challenges
  • the calculated cytotoxicity of the engineered cells having a tandem CAR with V H Hs connected by SEQ ID NO: 100 is higher (e.g., by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1 fold, at least 2 folds, at least 3 folds, at least 4 folds, at least 5 folds, at least 6 folds, at least 7 folds, at least 8 folds, at least 9 folds, at least 10 folds, at least 20 folds, at least 50 folds, or at least 100 folds) than that of the engineered cells having a CAR with V H Hs connected by SEQ ID NO: 99.
  • the engineered cells having a tandem CAR with V H Hs connected by SEQ ID NO: 100 have a maximum re-challenge number that is the same, at least 1 time, or at least 2 times more than that of the engineered cells having a CAR with V H Hs connected by SEQ ID NO: 99.
  • population of the engineered cells having a tandem CAR with V H Hs connected by SEQ ID NO: 100 is at least 1 fold, at least 2 folds, at least 3 folds, at least 4 folds, at least 5 folds, at least 6 folds, at least 7 folds, at least 8 folds, at least 9 folds, or at least 10 folds, as compared to that of the engineered cells having a CAR with V H Hs connected by SEQ ID NO: 99, after 1 re-challenge, 2 re-challenges, 3 re-challenges, 4 re-challenges, 5 re-challenges, or 6 re-challenges.
  • cytokine e.g., IFN- ⁇ , GM-CSF, and/or TNF- ⁇ expression or secretion of the engineered cells having a tandem CAR with V H Hs connected by SEQ ID NO: 100 is at least or about 100%, at least or about 110%, at least or about 120%, at least or about 130%as compared to that of the engineered cell having a CAR with V H Hs connected by SEQ ID NO: 99, after 1 re-challenge, 2 re-challenges, 3 re-challenges, 4 re-challenges, 5 re-challenges, or 6 re-challenges.
  • IFN- ⁇ e.g., IFN- ⁇ , GM-CSF, and/or TNF- ⁇
  • the cells are human PBMCs and engineered (e.g., transduced) to express the CAR, or antigen-binding fragment thereof.
  • the present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of recombinant polypeptides or fragments thereof by recombinant techniques.
  • recombinant vectors e.g., an expression vectors
  • an isolated polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleot
  • a vector is a construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell.
  • An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced.
  • the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-Atail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
  • regulatory elements such as a promoter, enhancer, and/or a poly-Atail
  • a vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) .
  • vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
  • the present disclosure provides a recombinant vector comprising a nucleic acid construct suitable for genetically modifying a cell, which can be used for treatment of pathological disease or condition.
  • Any vector or vector type can be used to deliver genetic material to the cell.
  • vectors include but are not limited to plasmid vectors, viral vectors, bacterial artificial chromosomes (BACs) , yeast artificial chromosomes (YACs) , and human artificial chromosomes (HACs) .
  • Viral vectors can include but are not limited to recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, foamy virus vectors, recombinant adeno-associated viral (AAV) vectors, hybrid vectors, and plasmid transposons (e.g., sleeping beauty transposon system, and PiggyBac transposon system) or integrase based vector systems.
  • AAV adeno-associated viral
  • Other vectors that are known in the art can also be used in connection with the methods described herein.
  • the vector is a viral vector.
  • the viral vector can be grown in a culture medium specific for viral vector manufacturing. Any suitable growth media and/or supplements for growing viral vectors can be used in accordance with the embodiments described herein.
  • the viral vector contains an EF1 ⁇ promoter to facilitate expression.
  • the vector used is a recombinant retroviral vector.
  • a retroviral vector is capable of directing the expression of a nucleic acid molecule of interest.
  • a retrovirus is present in the RNA form in its viral capsule and forms a double-stranded DNA intermediate when it replicates in the host cell.
  • retroviral vectors are present in both RNA and double-stranded DNA forms.
  • the retroviral vector also includes the DNA form which contains a recombinant DNA fragment and the RNA form containing a recombinant RNA fragment.
  • the vectors can include at least one transcriptional promoter/enhancer, or other elements which control gene expression.
  • Such vectors can also include a packaging signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites appropriate to the retrovirus used.
  • LTRs long terminal repeats
  • LTRs are identical sequences of DNA that repeat many times (e.g., hundreds or thousands of times) found at either end of retrotransposons or proviral DNA formed by reverse transcription of retroviral RNA. They are used by viruses to insert their genetic material into the host genomes.
  • the vectors can also include a signal which directs polyadenylation, selectable markers such as Ampicillin resistance, Neomycin resistance, TK, hygromycin resistance, phleomycin resistance histidinol resistance, or DHFR, as well as one or more restriction sites and a translation termination sequence.
  • retroviral vector used herein can also refers to the recombinant vectors created by removal of the retroviral gag, pol, and env genes and replaced with the gene of interest.
  • the vector or construct can contain a single promoter that drives the expression of one or more nucleic acid molecules.
  • promoters can be multicistronic (bicistronic or tricistronic) .
  • transcription units can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site) , which allows coexpression of gene products (e.g., encoding CAR and an antibody or antigen binding fragment thereof) by a message from a single promoter.
  • IRES internal ribosome entry site
  • a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF) , two or three genes (e.g., encoding CAR and/or an antibody or antigen binding fragment thereof) separated from one another by sequences encoding a self-cleavage peptide (e.g., P2A or T2A) or a protease recognition site (e.g., furin) .
  • ORF open reading frame
  • the ORF thus encodes a single polyprotein, which, either during (in the case of 2A e.g., T2A) or after translation, is cleaved into the individual proteins.
  • the peptide such as T2A
  • T2A can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream.
  • eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; HEK293 cells, including HEK293-6E cells; CHO cells, including CHO-S, DG44. Lec13 CHO cells, and FUT8 CHO cells; cells; and NSO cells.
  • a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the antibodies or CAR molecule.
  • CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in HEK293 cells.
  • the disclosure relates to a cell comprising the vector or the pair of vectors as described herein.
  • vectors encoding antibodies, CARs or fragments thereof.
  • the vectors comprise a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 73, 74, 75, 76, 77, 78, 79, 80, or 81.
  • the vectors encode an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98.
  • sequence of the vectors are codon-optimized.
  • nucleic acid sequence comprising a nucleotide sequence encoding any of the antibodies, CAR, antigen binding fragments thereof, and/or CAR-derived binding molecules (including e.g., functional portions and functional variants thereof, polypeptides, or proteins described herein) .
  • Nucleic acid as used herein can include “polynucleotide, ” “oligonucleotide, ” and “nucleic acid molecule, ” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained from natural sources, which can contain natural, non-natural or altered nucleotides.
  • the nucleic acid comprises complementary DNA (cDNA) . It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, it can be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and/or substitutions.
  • nucleic acids as described herein can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art.
  • a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides.
  • the nucleotide sequence is codon-optimized.
  • the present disclosure also provides the nucleic acids comprising a nucleotide sequence complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.
  • the nucleotide sequence encoding the CARs are separated by a peptide sequence that causes ribosome skipping.
  • the peptide that causes ribosome skipping is a P2A or T2A peptide.
  • the nucleic acid is synthetic. In some embodiments, the nucleic acid is cDNA.
  • the polypeptide comprises a signal peptide.
  • the signal peptide comprises a sequence that is at least at least 80%, at least 85%, at least 90%, at least 95%, or 100%identical to SEQ ID NO: 104.
  • the disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any amino acid sequence as described herein.
  • the disclosure relates to nucleotide sequence that is
  • the nucleic acid sequence is at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides.
  • the amino acid sequence is at least or about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, or 900 amino acid residues.
  • the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides.
  • the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, or 900 amino acid residues.
  • polynucleotide e.g., a vector
  • a schematic diagram of the polynucleotide is shown in FIG. 3, FIG. 4A, or FIG. 4B.
  • a schematic diagram of the polynucleotide (e.g., a vector) is shown in FIG. 4C.
  • the polynucleotide comprises, preferably from 5’ end to 3’ end, a sequence encoding a first GPC3-binding domain, a sequence encoding the hinge region (e.g., any of the hinge region derived from CD8 and/or CD28 disclosed herein) , a sequence encoding the transmembrane region (e.g., any of the transmembrane region derived from CD8 and/or CD28 disclosed herein) , a sequence encoding a first co-stimulatory domain, a sequence encoding the activating cytoplasmic signaling domain (e.g., any of the activating cytoplasmic signaling domain disclosed herein) , a self-cleave peptide (e.g., P2A or T2A) , a sequence encoding a second GPC3-binding domain
  • any one of the first CAR and second CAR further comprises a signal peptide and/or a tag.
  • the first CAR and second CAR both comprise a signal peptide and/or a tag.
  • the first and second signal peptides are the same.
  • the first and second signal peptides are different.
  • the tag is Flag tag.
  • the first and second tags are different.
  • the first and second GPC3-binding domains are the same.
  • the first and second GPC3-binding domains are different.
  • the first and second GPC3-binding domains are selected from any of the V H H combinations shown in Table 1.
  • the first and second co-stimulatory domains are the same. In some embodiments, the first and second co-stimulatory domains are different.
  • the present disclosure provides a method or process for preparing, manufacturing and/or using the engineered cells for treatment of pathological diseases or conditions.
  • the cells for introduction of the protein described herein, e.g., CAR can be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
  • a sample such as a biological sample, e.g., one obtained from or derived from a subject.
  • the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered.
  • the subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
  • the cells in some embodiments are primary cells, e.g., primary human cells.
  • the samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with viral vector) , washing, and/or incubation.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed.
  • Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
  • the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is derived from an apheresis or leukapheresis product.
  • exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs) , leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom.
  • Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
  • the cells are derived from cell lines, e.g., T cell lines.
  • the cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, or non-human primate. In some embodiments, the cells are isolated from mouse lymph nodes.
  • the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS) .
  • the wash solution lacks calcium and/or magnesium and/or many or all divalent cations.
  • a washing step is accomplished a semi-automated "flow-through” centrifuge.
  • a washing step is accomplished by tangential flow filtration (TFF) .
  • the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca 2+ /Mg 2+ free PBS.
  • components of a blood cell sample are removed and the cells directly resuspended in culture media.
  • the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
  • the method comprises one or more steps of: e.g., isolating the T cells from a patient’s blood; transducing the population T cells with a viral vector including the nucleic acid construct encoding a genetically engineered antigen receptor; expanding the transduced cells in vitro; and/or infusing the expanded cells into the patient, where the engineered T cells will seek and destroy antigen positive tumor cells.
  • the method further comprises: transfection of T cells with the viral vector containing the nucleic acid construct.
  • the methods involve introducing any vectors described herein into a cell in vitro or ex vivo.
  • the vector is a viral vector and the introducing is carried out by transduction.
  • the cell is transduced for at least or about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or longer.
  • the methods further involve introducing into the cell one or more agent, wherein each of the one or more agent is independently capable of inducing a genetic disruption of a T cell receptor alpha constant (TRAC) gene and/or a T cell receptor beta constant (TRBC) gene.
  • T cell receptor alpha constant TRAC
  • TRBC T cell receptor beta constant
  • the one or more agent is an inhibitory nucleic acid (e.g., siRNA) .
  • the one or more agent is a fusion protein comprising a DNA-targeting protein and a nuclease or an RNA-guided nuclease (e.g., a clustered regularly interspaced short palindromic nucleic acid (CRISPR) -associated nuclease) .
  • CRISPR clustered regularly interspaced short palindromic nucleic acid
  • transfection of T cells can be achieved by using any standard method such as calcium phosphate, electroporation, liposomal mediated transfer, microinjection, biolistic particle delivery system, or any other known methods by skilled artisan.
  • transfection of T cells is performed using the calcium phosphate method.
  • the present disclosure provides a method to create a personalized anti-tumor immunotherapy.
  • Genetically engineered T cells can be produced from a patient’s blood cells. These engineered T cells are then reinfused into the patient as a cellular therapy product.
  • the disclosure provides methods for treating a cancer in a subject, methods of reducing the rate of the increase of volume of a tumor in a subject over time, methods of reducing the risk of developing a metastasis, or methods of reducing the risk of developing an additional metastasis in a subject.
  • the treatment can halt, slow, retard, or inhibit progression of a cancer.
  • the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the cancer in a subject.
  • the disclosure features methods that include administering a therapeutically effective amount of antibodies or antigen binding fragments thereof, or engineered cells expressing CAR, to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a cancer) .
  • the subject has GPC3-positive cancer.
  • the subject has liver cancer (e.g., hepatocellular carcinoma) , glioma, lung cancer, colorectal cancer, head and neck cancer, stomach cancer, renal cancer, urothelial cancer, testis cancer, breast cancer, cervical cancer, endometrial cancer, and/or ovarian cancer.
  • the subject has squamous cell lung carcinoma, or solid tumor.
  • the subject has a CNS tumor, thyroid cancer, gastrointestinal cancer, skin cancer, sarcoma, urogenital cancer, and/or germ cell tumor.
  • GPC3-related cancers can be found, e.g., in Moek, Kirsten L., et al., The American Journal of Pathology 188.9 (2016) : 1973-1981, which is incorporated herein by reference in its entirety.
  • compositions and methods disclosed herein can be used for treatment of patients at risk for a cancer.
  • Patients with cancer can be identified with various methods known in the art.
  • an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a cancer.
  • An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the therapeutic agent and/or therapeutic compositions is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.
  • the term "delaying development of a disease” refers to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer) .
  • This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated.
  • a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.
  • a late stage cancer such as development of metastasis, can be delayed.
  • an effective amount can be administered in one or more administrations.
  • an effective amount of a composition is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of a cancer in a patient or is an amount sufficient to ameliorate, stop, stabilize, reverse, slow and/or delay proliferation of a cell (e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line) ) in vitro.
  • a cell e.g., a biopsied cell, any of the cancer cells described herein, or cell line (e.g., a cancer cell line)
  • an effective may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of compositions used.
  • Effective amounts and schedules for administrations may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the treatment, the route of administration, the particular type of therapeutic agents and other drugs being administered to the mammal. Guidance in selecting appropriate doses can be found in the literature. In addition, a treatment does not necessarily result in the 100%or complete treatment or prevention of a disease or a condition. There are multiple treatment/prevention methods available with a varying degree of therapeutic effect which one of ordinary skill in the art recognizes as a potentially advantageous therapeutic mean.
  • the present disclosure also provides methods of diagnosing a disease/condition in a mammal, wherein the CARs, antibodies, or antigen binding fragments, interact with the sample (s) obtained from a subject to form a complex, wherein the sample can comprise one more cells, polypeptides, proteins, nucleic acids, antibodies, or antigen binding portions, blood, whole cells, lysates thereof, or a fraction of the whole cell lysates, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction thereof, wherein the detection of the complex is the indicative of presence of a condition in the mammal, wherein the condition is cancer or infection.
  • the sample can comprise one more cells, polypeptides, proteins, nucleic acids, antibodies, or antigen binding portions, blood, whole cells, lysates thereof, or a fraction of the whole cell lysates, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic
  • the detection of the complex can be in any number of ways known in the art but not limited to, ELISA, Flow cytometery, Fluorescence in situ hybridization (FISH) , Polymerase chain reaction (PCR) , microarray, southern blotting, electrophoresis, Phage analysis, chromatography and more.
  • the treatment methods can further include determining whether a subject can benefit from a treatment as disclosed herein, e.g., by determining whether the subject has infection or cancer.
  • the engineered cells can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) .
  • at least two different engineered cells e.g., cells expressing different CARs
  • engineered cells and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition) .
  • engineered cells and at least one additional therapeutic agent are administered in two different compositions.
  • the at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained-release oral formulation. In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, concurrently with, or after administering the engineered cells to the subject.
  • the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of B-Raf, an EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of anaplastic lymphoma kinase (ALK) , an inhibitor of a phosphatidylinositol 3-kinase (PI3K) , an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K/mTOR inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK) , and an inhibitor of Isocitrate dehydrogenase 1 (IDH1) and/or Isocitrate dehydrogenase 2 (IDH2) .
  • an inhibitor of B-Raf an EGFR inhibitor
  • an inhibitor of a MEK an inhibitor of ERK
  • K-Ras an inhibitor of c-Met
  • ALK an inhibitor
  • the additional therapeutic agent is an inhibitor of indoleamine 2, 3-dioxygenase-1) (IDO1) (e.g., epacadostat) .
  • the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of an inhibitor of HER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades the estrogen receptor.
  • the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, Temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, prala
  • therapeutic agents
  • the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and a Selectin agonist.
  • TNF tumor necrosis factor
  • carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.
  • the additional therapeutic agent is selected from asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine and/or combinations thereof.
  • compositions including pharmaceutical and therapeutic compositions
  • methods e.g., therapeutic methods for administrating the engineered cells and compositions thereof to subjects, e.g., patients or animal models (e.g., mice) .
  • compositions including the engineered cells for administration including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof are provided.
  • the pharmaceutical compositions and formulations can include one or more optional pharmaceutically acceptable carrier or excipient.
  • the composition includes at least one additional therapeutic agent.
  • a pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition, other than an active ingredient.
  • the pharmaceutically acceptable carrier does not interfere with the active ingredient and is nontoxic to a subject.
  • a pharmaceutically acceptable carrier can include, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • the pharmaceutical formulation refers to process in which different substances and/or agents are combined to produce a final medicinal product. The formulation studies involve developing a preparation of drug acceptable for patient. Additionally, a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • the choice of carrier is determined in part by the particular cell (e.g., T cell or NK cell) and/or by the method of administration.
  • the pharmaceutical composition can contain preservatives. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001%to about 2%by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) .
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
  • Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001%to about 4%by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005) .
  • the formulations can include aqueous solutions.
  • the formulation or composition can also contain more than one active ingredient useful for a particular indication, disease, or condition being treated with the engineered cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • the pharmaceutical composition can further include other pharmaceutically active agents or drugs, such as checkpoint inhibitors, fusion proteins, chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
  • other pharmaceutically active agents or drugs such as checkpoint inhibitors, fusion proteins, chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and/or vincristine.
  • chemotherapeutic agents e.g., asparaginase
  • the pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount.
  • Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects.
  • the desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.
  • the cells and compositions can be administered using standard administration techniques, formulations, and/or devices. Administration of the cells can be autologous or heterologous.
  • immunoresponsive T cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject after genetically modifying them in accordance with various embodiments described herein.
  • Peripheral blood derived immunoresponsive T cells or their progeny e.g., in vivo, ex vivo or in vitro derived
  • a therapeutic composition e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell
  • it is generally formulated in a unit dosage injectable form (solution, suspension, emulsion) .
  • Formulations disclosed herein include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
  • the cell populations are administered parenterally.
  • parenteral, ” as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration.
  • the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
  • Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.
  • a suitable carrier such as a suitable carrier, diluent, or excipient
  • the compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose) , pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and/or colors, depending upon the route of administration and the preparation desired. Standard texts can in some aspects be consulted to prepare suitable preparations.
  • compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
  • antimicrobial preservatives for example, parabens, chlorobutanol, phenol, and sorbic acid.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the formulations to be used for in vivo administration are generally sterile. Sterility can be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • compositions or pharmaceutical compositions as described herein can be included in a container, pack, or dispenser together with instructions for administration.
  • the methods described herein can reduce the risk of the developing diseases, conditions, and disorders as described herein.
  • the cells, populations, and compositions, described herein are administered to a subject or patient having a particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy.
  • adoptive cell therapy such as adoptive T cell therapy.
  • cells and compositions prepared by the provided methods such as engineered compositions and end-of-production compositions following incubation and/or other processing steps, are administered to a subject, such as a subject having or at risk for the disease or condition.
  • the methods thereby treat, e.g., ameliorate one or more symptom of, the disease or condition, such as by lessening tumor burden in cancer expressing an antigen recognized by the engineered T cells.
  • the cell therapy e.g., adoptive T cell therapy
  • the cell therapy is carried out by autologous transfer, in which the T cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject.
  • the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
  • the cell therapy e.g., adoptive T cell therapy
  • the cell therapy is carried out by allogeneic transfer, in which the T cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject.
  • the cells then are administered to a different subject, e.g., a second subject, of the same species.
  • the first and second subjects are genetically identical.
  • the first and second subjects are genetically similar.
  • the second subject expresses the same HLA class or supertype as the first subject.
  • the subject has been treated with a therapeutic agent targeting the disease or condition, e.g., the tumor, prior to administration of the cells or composition containing the cells.
  • a therapeutic agent targeting the disease or condition, e.g., the tumor, prior to administration of the cells or composition containing the cells.
  • the subject is refractory or non-responsive to the other therapeutic agent.
  • the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT) , e.g., allogenic HSCT.
  • the administration effectively treats the subject despite the subject having become resistant to another therapy.
  • the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden.
  • the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time.
  • the subject has not relapsed.
  • the subject is determined to be at risk for relapse, such as at high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.
  • the subject has not received prior treatment with another therapeutic agent.
  • the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type (s) and/or a desired ratio of cell types.
  • the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio.
  • the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types.
  • the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.
  • the populations or sub-types of cells are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells.
  • the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg.
  • the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight.
  • the individual populations or sub-types are present at or near a desired output ratio (such as CD4+ to CD8+ ratio) , e.g., within a certain tolerated difference or error of such a ratio.
  • a desired output ratio such as CD4+ to CD8+ ratio
  • the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired dose of CD4+ cells and/or a desired dose of CD8+ cells.
  • the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg.
  • the desired dose is at or above a minimum number of cells of the population or sub-type, or minimum number of cells of the population or sub-type per unit of body weight.
  • the dosage is based on a desired fixed dose of total cells and a desired ratio, and/or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or sub-populations.
  • the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4+ to CD8+ cells, and/or is based on a desired fixed or minimum dose of CD4+ and/or CD8+ cells.
  • the cells or individual populations of sub-types of cells are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values) , such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values) , and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells,
  • the dose of total cells and/or dose of individual sub-populations of cells is within a range of between at or about 10 4 and at or about 10 9 cells/kilograms (kg) body weight, such as between 10 5 and 10 6 cells/kg body weight, for example, at least or at least about or at or about 1 ⁇ 10 5 cells/kg, 1.5 ⁇ 10 5 cells/kg, 2 ⁇ 10 5 cells/kg, or 1 ⁇ 10 6 cells/kg body weight.
  • the cells are administered at, or within a certain range of error of, between at or about 10 4 and at or about 10 9 T cells/kilograms (kg) body weight, such as between 10 5 and 10 6 T cells/kg body weight, for example, at least or at least about or at or about 1 ⁇ 10 5 T cells/kg, 1.5 ⁇ 10 5 T cells/kg, 2 ⁇ 10 5 T cells/kg, or 1 ⁇ 10 6 T cells/kg body weight.
  • the cells are administered at or within a certain range of error of between at or about 10 4 and at or about 10 9 CD4+ and/or CD8+ cells/kilograms (kg) body weight, such as between 10 5 and 10 6 CD4+ and/or CD8+ cells/kg body weight, for example, at least or at least about or at or about 1 ⁇ 10 5 CD4+ and/or CD8+ cells/kg, 1.5 ⁇ 10 5 CD4+ and/or CD8+ cells/kg, 2 ⁇ 10 5 CD4+ and/or CD8+ cells/kg, or 1 ⁇ 10 6 CD4+ and/or CD8+ cells/kg body weight.
  • body weight such as between 10 5 and 10 6 CD4+ and/or CD8+ cells/kg body weight, for example, at least or at least about or at or about 1 ⁇ 10 5 CD4+ and/or CD8+ cells/kg, 1.5 ⁇ 10 5 CD4+ and/or CD8+ cells/kg, 2 ⁇ 10 5 CD4+ and/or CD8+ cells/kg, or 1 ⁇ 10 6 CD4+ and
  • the cells are administered at or within a certain range of error of, greater than, and/or at least about 1 ⁇ 10 6 , about 2.5 ⁇ 10 6 , about 5 ⁇ 10 6 , about 7.5 ⁇ 10 6 , or about 9 ⁇ 10 6 CD4+ cells, and/or at least about 1 ⁇ 10 6 , about 2.5 ⁇ 10 6 , about 5 ⁇ 10 6 , about 7.5 ⁇ 10 6 , or about 9 ⁇ 10 6 CD8+ cells, and/or at least about 1 ⁇ 10 6 , about 2.5 ⁇ 10 6 , about 5 ⁇ 10 6 , about 7.5 ⁇ 10 6 , or about 9 ⁇ 10 6 T cells.
  • the cells are administered at or within a certain range of error of between about 10 8 and 10 12 or between about 10 10 and 10 11 T cells, between about 10 8 and 10 12 or between about 10 10 and 10 11 CD4+ cells, and/or between about 10 8 and 10 12 or between about 10 10 and 10 11 CD8+ cells.
  • the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4+ and CD8+ cells or sub-types.
  • the desired ratio can be a specific ratio or can be a range of ratios.
  • the desired ratio (e.g., ratio of CD4+ to CD8+ cells) is between at or about 1: 5 and at or about 5: 1 (or greater than about 1: 5 and less than about 5: 1) , or between at or about 1: 3 and at or about 3: 1 (or greater than about 1: 3 and less than about 3: 1) , such as between at or about 2: 1 and at or about 1: 5 (or greater than about 1: 5 and less than about 2: 1, such as at or about 5: 1, 4.5: 1, 4: 1, 3.5: 1, 3: 1, 2.5: 1, 2: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1, 1.2: 1, 1.1: 1, 1: 1, 1: 1.1, 1: 1.2, 1: 1.3, 1: 1.4, 1: 1.5, 1: 1.6, 1: 1.7, 1: 1.8, 1: 1.9: 1: 2, 1: 2.5, 1: 3, 1: 3.5, 1: 4, 1: 4.5
  • the tolerated difference is within about 1%, about 2%, about 3%, about 4%about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%of the desired ratio, including any value in between these ranges.
  • the CAR described here provides improved expression and activity, thereby providing therapeutic effects even at a low effector to target (E: T) ratio.
  • Optimal response to therapy can depend on the ability of the engineered recombinant receptors such as CARs, to be consistently and reliably expressed on the surface of the cells and/or bind the target antigen.
  • properties of certain recombinant receptors e.g., CARs
  • CARs can affect the expression and/or activity of the recombinant receptor, in some cases when expressed in a cell, such as a human T cell, used in cell therapy.
  • the level of expression of particular recombinant receptors, e.g., CARs can be low, and activity of the engineered cells, such as human T cells, expressing such recombinant receptors, may be limited due to poor expression or poor signaling activity.
  • the desired ratio is between at or about 1: 10 and at or about 10: 1 (or greater than about 1: 10 and less than about 10: 1) , or between at or about 1: 1 and at or about 10: 1 (or greater than about 1: 1 and less than about 5: 1) , such as between at or about 2: 1 and at or about 10: 1.
  • the E: T ratio is greater than or about 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1. In some embodiments, the E: T ratio is about 3: 1, about 1: 1, or about 0.3: 1.
  • the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician.
  • the compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.
  • the cells described herein can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • injection e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • injection e.g., intravenous or
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • a given dose is administered by a single bolus administration of the cells. In some embodiments, it is administered by multiple bolus administrations of the cells, for example, over a period of no more than 3 days, or by continuous infusion administration of the cells.
  • the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent.
  • the cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order.
  • the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa.
  • the cells are administered prior to the one or more additional therapeutic agents.
  • the cells are administered after the one or more additional therapeutic agents.
  • the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence.
  • the methods comprise administration of a chemotherapeutic agent.
  • the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods.
  • Parameters to assess include specific binding of engineered T cells to the antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry.
  • the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., "Construction and pre-clinical evaluation of an anti-CD19 chimeric antigen receptor.
  • the biological activity of the cells is measured by assaying expression and/or secretion of one or more cytokines, such as CD107a, IFN ⁇ , IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
  • Repeated dosing methods are provided in which a first dose of cells is given followed by one or more second consecutive doses.
  • the timing and size of the multiple doses of cells generally are designed to increase the efficacy and/or activity and/or function of engineered cells as described herein, when administered to a subject in adoptive therapy methods.
  • the methods involve administering a first dose, generally followed by one or more consecutive doses, with particular time frames between the different doses.
  • administration of a given “dose” encompasses administration of the given amount or number of cells as a single composition and/or single uninterrupted administration, e.g., as a single injection or continuous infusion, and also encompasses administration of the given amount or number of cells as a split dose, provided in multiple individual compositions or infusions, over a specified period of time (e.g., no more than 3 days) .
  • the first or consecutive dose is a single or continuous administration of the specified number of cells, given or initiated at a single point in time.
  • the first or consecutive dose is administered in multiple injections or infusions over a limited time period (e.g., no more than three days) , such as once a day for three days or for two days or by multiple infusions over a single day period.
  • a limited time period e.g., no more than three days
  • the cells of the first dose are administered in a single pharmaceutical composition.
  • the cells of the consecutive dose are administered in a single pharmaceutical composition.
  • the cells of the first dose are administered in a plurality of compositions, collectively containing the cells of the first dose.
  • the cells of the consecutive dose are administered in a plurality of compositions, collectively containing the cells of the consecutive dose.
  • additional consecutive doses can be administered in a plurality of compositions over a period of no more than 3 days.
  • the term “consecutive dose” refers to a dose that is administered to the same subject after the prior, e.g., first, dose without any intervening doses having been administered to the subject in the interim. Nonetheless, the term does not encompass the second, third, and/or so forth, injection or infusion in a series of infusions or injections comprised within a single split dose. Thus, unless otherwise specified, a second infusion within a one, two or three-day period is not considered to be a “consecutive” dose as used herein.
  • a second, third, and so-forth in the series of multiple doses within a split dose also is not considered to be an “intervening” dose in the context of the meaning of “consecutive” dose.
  • a dose administered a certain period of time, greater than three days, after the initiation of a first or prior dose is considered to be a “consecutive” dose even if the subject receives a second or subsequent injection or infusion of the cells following the initiation of the first dose, so long as the second or subsequent injection or infusion occurred within the three-day period following the initiation of the first or prior dose.
  • multiple administrations of the same cells over a period of up to 3 days is considered to be a single dose, and administration of cells within 3 days of an initial administration is not considered a consecutive dose and is not considered to be an intervening dose for purposes of determining whether a second dose is “consecutive” to the first.
  • multiple consecutive doses are given, in some aspects using the same timing guidelines as those with respect to the timing between the first dose and first consecutive dose, e.g., by administering a first and multiple consecutive doses.
  • the timing between the first dose and first consecutive dose, or a first and multiple consecutive doses is such that each consecutive dose is given within a period of time is greater than about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days or more.
  • the consecutive dose is given within a time period that is less than about 28 days after the administration of the first or immediately prior dose.
  • the additional multiple additional consecutive dose or doses also are referred to as subsequent dose or subsequent consecutive dose.
  • the size of the first and/or one or more consecutive doses of cells are generally designed to provide improved efficacy and/or reduced risk of toxicity.
  • a dosage amount or size of a first dose or any consecutive dose is any dosage or amount as described above.
  • the number of cells in the first dose or in any consecutive dose is between about 0.5 ⁇ 10 6 cells/kg body weight of the subject and 5 ⁇ 10 6 cells/kg, between about 0.75 ⁇ 10 6 cells/kg and 3 ⁇ 10 6 cells/kg or between about 1 ⁇ 10 6 cells/kg and 2 ⁇ 10 6 cells/kg.
  • first dose is used to describe the timing of a given dose being prior to the administration of a consecutive or subsequent dose. The term does not necessarily imply that the subject has never before received a dose of cell therapy or even that the subject has not before received a dose of the same cells or cells expressing the same recombinant receptor or targeting the same antigen.
  • multiple doses can be administered to a subject over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years) .
  • a skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of cancer) .
  • An immunogen comprising a recombinant human GPC3 protein having a C-terminal His tag was mixed with adjuvant or PBS, and then was injected to llamas.
  • the animals were immunized for seven times, typically with 200 ⁇ g immunogen and CFA (Complete Freund's Adjuvant) each time at about 1-week to 2-week intervals.
  • CFA Complete Freund's Adjuvant
  • Peripheral blood samples were collected at the pre-immunization stage and after the 3rd, 5th and 7th immunization. After multiple rounds of immunization, immune reactions against the target antigen were evaluated to confirm the titer of antigen-specific binders. As shown in FIGS.
  • Peripheral blood lymphocytes PBLs
  • camel HcAbs camel HcAbs (heavy-chain only antibodies)
  • the cells were supplemented with RNAlaterTM solutions and stored at -80 °C.
  • Sera were obtained by centrifugation of anti-coagulated blood samples and were stored at -80 °C.
  • RNA extracted from PBLs was used as starting material for RT-PCR to amplify sdAb-encoding gene fragments. These fragments were cloned into an in-house phagemid vector. The library size was about 1.5 ⁇ 10 9 . Phage libraries were prepared according to standard protocols and stored after filter-sterilization at 4 °C for further use.
  • the phage libraries were screened by solid panning as well as liquid panning. Only a single round of selection was performed for each of the two panning conditions. Each selection output was analyzed to determine an enrichment factor (i.e., number of phages present in the eluate relative to control) , diversity and percentage of GPC3-positive clones based on ELISA results. Based on these parameters, the best clones were chosen for further analysis. To this end, the output from each selection was re-cloned as a pool into a soluble expression vector for high-throughput screening.
  • an enrichment factor i.e., number of phages present in the eluate relative to control
  • the supernatants from 64 clones were first screened for their ability to bind recombinant human and cynomolgus monkey GPC3 proteins using an ELISA assay.
  • the positive binders were sequenced and unique clones were selected for further characterization.
  • the supernatants of unique binders were analyzed for their ability to bind to GPC3-expressing CHO-3E7/human GPC3 cell line, CHO-3E7/monkey (Rhesus) GPC3 cell line and cancer cell line HepG2 using a fluorescence-activated cell sorting (FACS) assay. Binding data of selected anti-GPC3 binders are shown in the table below.
  • each isolated sdAb binder (SEQ ID NOs: 64-72) was cloned into a lentiviral expression vector with the intracellular co-stimulatory sequence of 4-1BB and intracellular domain of CD3 ⁇ as shown in FIG. 3 and the table below.
  • the constructs encoding CAR (SEQ ID NOs: 82-90) were cloned into an expression vector with an EF1 ⁇ promoter to facilitate expression.
  • a construct encoding anti-GPC3 benchmark CAR ( “BMCAR” ) was also prepared for comparative analysis using the sequence shown in FIG. 17 (SEQ ID NO: 112) .
  • the lentivirus packaging plasmid mixture including pCMV- ⁇ R-8.47 and pMD2. G (Addgene, Cat#12259) , was pre-mixed with each vector PLLV-hEF1 ⁇ -GPC3 comprising a CAR construct at a pre-optimized ratio with polyethylenimine. The mixture was then added to HEK293 cells. Supernatants from the cells were collected after overnight incubation. The virus-containing supernatants were filtered through a 0.45 ⁇ m PES filter, followed by ultra-centrifugation to pellet the lentivirus. The virus pellets were rinsed with pre-chilled PBS. Afterwards, the virus was aliquoted and stored at -80 °C immediately. The virus titer was determined by measurement of transduction efficiency to supT1 cell line using a flow cytometry assay.
  • PBMCs Peripheral blood mononuclear cells
  • FICOLL-PAQUE TM PLUS Media GE Healthcare, Cat#17-5442-02
  • Human T cells were purified from PMBCs using a Pan-T cell isolation kit (Miltenyi, Cat#130-096-535) , following manufacturer’s protocol.
  • the purified T cells were subsequently pre-activated for 48 hours with a human T cell activation/expansion kit (Miltenyi, Cat#130-091-441) according to manufacturer’s protocol in which anti-CD3/CD28 MACSiBead particles were added at a bead-to-cell ratio of 1: 2.
  • the pre-activated T cells were transduced with each lentivirus stock in the presence of 7 ⁇ g/ml polybrene. The transduced cells were then transferred to a cell culture incubator for transgene expression under suitable conditions.
  • the positive rates of virus-infected T cells expressing different CARs are shown in the table below.
  • the viability and expansion of anti-GPC3 CAR-T cells with sdAbs are shown in FIGS. 5A-5B.
  • FACS assays were performed to determine cytotoxicity.
  • transduced T cells were harvested and co-incubated with target cells (GPC3 + HCC cell line: Huh7 and PLC-PRF5, GPC3 - HCC cell line: SK-HEP-1) at an E: T ratio (Effector (E) : CAR-T/Target (T) : Huh7, PLC-PRF5, or SK-HEP-1) of 3: 1, 1: 1, or 0.3: 1 for 16 hours.
  • the target cells were pre-stained for carboxyfluorescein succinimidyl ester (CFSE) .
  • Un-transduced T cells (UNT) from the same batch were used as a negative control.
  • GPC3 benchmark CAR-T cells ( “BMCAR” ) were used as positive control.
  • the mixed cells were harvested and stained with 7-aminoactinomycin D (7-ADD) .
  • the cytotoxicity was calculated using the equation below:
  • Cytotoxicity% (7AAD+ (E+T) -7AAD+ (T) ) / (1-7AAD+ (T) ) ⁇ 100
  • 7AAD+ (E+T) is the percentage of cells stained by 7-ADD in E: T cells (co-incubation)
  • 7AAD+ (T) is the percentage of cells stained by 7-ADD in target cells only.
  • E+T is the percentage of cells stained by 7-ADD in E: T cells (co-incubation)
  • 7AAD+ (T) is the percentage of cells stained by 7-ADD in target cells only.
  • FIGS. 6A-6C all anti-GPC3 CAR-T cells showed potent cytotoxicity against GPC3 + HCC cell line Huh7 and PLC-PRF5 cells. Cytotoxicity of all anti-GPC3 CAR-T cells was comparable to that of the BMCAR-T cells. In contrast, a significantly less GPC3 - HCC cell line SK-HEP-1 cells were killed after incubation with the anti-GPC3 CAR-T cells.
  • T cells Transduced or un-transduced T cells (1 ⁇ 10 5 /well) were co-cultured with tumor cell lines (Huh7 cells, 1 ⁇ 10 5 /well) at an E: T ratio of 1: 1 in 24-well plates, in the absence of exogenous cytokines (e.g., IL-2) . After co-culturing for 2 or 3 days, a portion of the cell pool was harvested and CD3 was stained. For serial co-culture assays, the remaining T cells were then re-challenged with fresh Huh7 cells at the same E: T ratio. The remaining T cells were then collected.
  • tumor cell lines Huh7 cells, 1 ⁇ 10 5 /well
  • E exogenous cytokines
  • the T cell proliferation rate at each time point is calculated by dividing the number of T cells at the time point by the number of T cells at the initial time point.
  • FIG. 7A A representative result of long-term co-culture assays by FACS detection is shown in FIG. 7A. Calculated T cell proliferation curves from the same experiment are shown in FIG. 7B. The results indicate that the CAR-T cells having CAR derived from sdAbs proliferated and persisted well.
  • IFN- ⁇ , GM-CSF, and TNF- ⁇ secretion detected by HTRF IFN- ⁇ , GM-CSF, and TNF- ⁇ secretion detected by HTRF
  • effector T cell activation and proliferation is the production of effector cytokines, e.g., IFN- ⁇ , GM-CSF, and TNF- ⁇ .
  • the first three rounds of supernatants from the in vitro cytotoxicity assays were collected to assess CAR-induced cytokine release.
  • Homogeneous time resolved fluorescence (HTRF) assays for IFN- ⁇ (Cisbio, Cat#62HIFNGPEH) , GM-CSF (Cisbio, Cat#62HGMCSFPEH) , and TNF ⁇ (Cisbio, Cat#62HTNFAPEH) were performed according to the manufacturer’s manual.
  • the corresponding cytokine release results are shown in FIGS. 8A-8C, respectively.
  • Exemplary tandem CARs as shown in FIG. 4B were constructed by fusing two binding domains specifically recognizing GPC3 via a peptide linker to form the extracellular domain in a single CAR molecule.
  • Anti-GPC3 tandem CARs were cloned into a lentiviral expression vector with the intracellular co-stimulatory sequence of 4-1BB and intracellular domain of CD3 ⁇ as shown in FIG. 4B and the table below.
  • the CAR constructs were cloned into an expression vector with an EF1 ⁇ promoter to facilitate expression.
  • T lymphocytes were collected and transduced with the lentiviruses according to the protocol in Example 2.
  • FIGS. 9A-9D The killing efficacy of various tandem CAR-T cells in the repeated tumor stimulation assay is shown in FIGS. 9A-9D.
  • linker 2 (GGGGSGGGGSGGGGS; SEQ ID NO: 100) was more conducive to the function of tandem CAR-T cells than linker 1 (GGGGS; SEQ ID NO: 99) .
  • the sequential position of the two sdAbs also influenced the function of tandem CAR-T cells.
  • FIGS. 10A-10B the results also showed that the tandem CAR-T cells had better cytotoxicity and proliferation than single CAR-T cells (CAR-T cells with the CAR structure having a single extracellular sdAb) in vitro.
  • IFN- ⁇ , GM-CSF and TNF- ⁇ secretion detected by HTRF IFN- ⁇ , GM-CSF and TNF- ⁇ secretion detected by HTRF
  • Cytokine release (IFN- ⁇ , GM-CSF, and TNF- ⁇ ) by tandem CAR-T cells in long-term co-cultures with tumor cells was assessed as described in Example 2.
  • Results of a representative cytokine release assay are shown in FIGS. 11A-11C. The results showed that the tandem CAR-T cells released higher levels of cytokines as compared to those released by single CAR-T cells, in the presence of Huh7 cells. Further, the results demonstrate that tandem CAR-T cells have more potent anti-tumor activity than single CAR-T cells in vitro.
  • mice were injected of 0.5 ⁇ 10 6 CAR-T cells, UNT cells, or Hank's Balanced Salt Solution (HBSS) by intravenous administration. Tumor dimensions were measured with calipers twice a week, and tumor volumes are calculated using the formula V 1/2 (length ⁇ width 2 ) . Mice were euthanized when the mean tumor size in the mice reached 2,000 mm 3 .
  • mice treated with un-transduced T cells (UNT) or vehicle exhibited rapid tumor progression and had to be euthanized before the end of the experiment.
  • mice treated with Tan6 CAR-T cells were tumor free after about 3-4 weeks post injection, and exhibited much slower tumor growth than mice treated with BMCAR-T cells. The result showed that tumor growth in mice treated with tandem CAR-T cells was efficacy in vivo.
  • Exemplary dual CARs as shown in FIG. 4C were constructed by expressing two fully functional CARs against GPC3.
  • the dual CAR constructs were cloned into an expression vector with an EF1 ⁇ promoter to facilitate expression.
  • Lentiviruses encoding the dual CAR constructs as well as the individual CARs contained therein were prepared. T lymphocytes were collected and transduced with the lentiviruses according to the protocol in Example 2.

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Abstract

L'invention concerne des anticorps à domaine unique ciblant GPC3, et des récepteurs antigéniques chimériques (par exemple, CAR monovalent, et CAR multivalent comprenant CAR bi-épitope) ayant un ou plusieurs anticorps à domaine unique anti-GPC3. L'invention concerne en outre des cellules effectrices immunitaires modifiées (par exemple, des lymphocytes T) exprimant les récepteurs antigéniques chimériques et leurs procédés d'utilisation.
PCT/CN2022/090894 2021-04-30 2022-05-05 Récepteurs antigéniques chimériques ciblant gpc3 et leurs procédés d'utilisation WO2022228579A1 (fr)

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WO2024118866A1 (fr) * 2022-12-01 2024-06-06 Modernatx, Inc. Anticorps spécifiques au gpc3, domaines de liaison, protéines associées et leurs utilisations

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US20200048359A1 (en) * 2017-02-28 2020-02-13 Novartis Ag Shp inhibitor compositions and uses for chimeric antigen receptor therapy
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WO2024118866A1 (fr) * 2022-12-01 2024-06-06 Modernatx, Inc. Anticorps spécifiques au gpc3, domaines de liaison, protéines associées et leurs utilisations

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