Patent Application
20220169710
The present disclosure relates to an antibody or antigen-binding fragment thereof, which is specific to stage-specific embryonic antigen 4 (SSEA4) for enrichment immune cells.
Cell therapy, also known as cellular therapy, is developed as a powerful strategy to treat many diseases such as cancers, genetic diseases, and autoimmune disorders. The therapy comprises introducing desired cells into a patient in need of such treatment for exerting a medicinal effect. Among various cells utilized in cell therapy, immune cells play an important role in view of their outstanding success in cancer treatment. For example, transplanting T-cells is capable of inhibiting cancer growth via cell-mediated immunity. Enrichment of desired immune cells is essential in the field of cell therapy. Conventional methods for selecting desired immune cells among peripheral blood mononuclear cells (PBMCs) usually utilize an antibody specific to a unique antigen located on the surface of the desired immune cells. For example, an anti-CD56 antibody is applied in selecting natural killer cells which express CD56 antigen on the cell surface. However, almost natural killer cells express CD56 antigen, and the conventional method fails to select sub-groups among killer cells.
Thus, there is need for developing a novel approach to enrichment of cells.
The present disclosure relates to a method for enrichment of cells with the modified antibody or antigen-binding fragment thereof The present disclosure also provides an antibody or antigen-binding fragment thereof that specifically binds to an epitope in an antigen or a fragment thereof.
In one aspect, the present disclosure provides an the antibody or antigen-binding fragment thereof comprising complementarity determining regions (CDRs) of a heavy chain variable region and CDRs of a light chain variable region, wherein the CDRs of the heavy chain variable region comprises CDRH1, CDRH2 and CDRH3 regions, and the CDRs of the light chain variable region comprises CDRL1, CDRL2 and CDRL3 regions, and wherein:
In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof or Fc-fused antigen-binding fragment thereof comprising the CDRs as disclosed herein.
In one embodiment of the disclosure, the CDRH1 region comprises the amino acid sequence of SEQ ID NO: 7, 13 or 14.
In one embodiment of the disclosure, the CDRH2 region comprises the amino acid sequence of SEQ ID NO: 8 or 15.
In one embodiment of the disclosure, the CDRH3 region comprises the amino acid sequence of SEQ ID NO: 9, 16, 17, or 18.
In one embodiment of the disclosure, the CDRL1 region comprises the amino acid sequence of SEQ ID NO: 10 or 19.
In one embodiment of the disclosure, the CDRL2 region comprises the amino acid sequence of SEQ ID NO: 11, 20 or 21.
In one embodiment of the disclosure, the CDRL3 region comprises the amino acid sequence of SEQ ID NO: 12 or 22.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 13, the CDRH2 region being SEQ ID NO: 8, the CDRH3 region being SEQ ID NO: 9, the CDRL1 region being SEQ ID NO: 10, the CDRL2 region being SEQ ID NO: 20 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 13, the CDRH2 region SEQ ID NO: 15, the CDRH3 region being SEQ ID NO: 9, the CDRL1 region being SEQ ID NO: 10, the CDRL2 region being SEQ ID NO: 11 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 13, the CDRH2 region being SEQ ID NO: 8, the CDRH3 region being SEQ ID NO: 16, the CDRL1 region being SEQ ID NO: 10, the to CDRL2 region being SEQ ID NO: 11 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 14, the CDRH2 region being SEQ ID NO: 8, the CDRH3 region being SEQ ID NO: 17, the CDRL1 region being SEQ ID NO: 10, the CDRL2 region being SEQ ID NO: 21 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 14, the CDRH2 region being SEQ ID NO: 8, the CDRH3 region being SEQ ID NO: 18, the CDRL1 region being SEQ ID NO: 10, the CDRL2 region being SEQ ID NO: 11 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 7, the CDRH2 region being SEQ ID NO: 15, the CDRH3 region being SEQ ID NO: 9, the CDRL1 region being SEQ ID NO: 10, the CDRL2 region being SEQ ID NO: 20 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 7, the CDRH2 region being SEQ ID NO: 15, the CDRH3 region being SEQ ID NO: 17, the CDRL1 region being SEQ ID NO: 10, the CDRL2 region being SEQ ID NO: 11 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 7, the CDRH2 region being SEQ ID NO: 8, the CDRH3 region being SEQ ID NO: 16, the CDRL1 region being SEQ ID NO: 10, the CDRL2 region being SEQ ID NO: 20 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 or a substantially similar sequence thereof and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24 or a substantially similar sequence thereof.
In one embodiment of the disclosure, the antigen-binding fragment is a single-chain FAT (scFv) molecule comprising the amino acid sequence of SEQ ID NO: 37.
In one embodiment of the disclosure, the antigen-binding fragment is fused to an Fc region having the amino acid sequence of SEQ ID NO: 38.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33 or a substantially similar sequence thereof; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 34 or a substantially similar sequence thereof.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof further comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 35 or a substantially similar sequence thereof; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 36 or a substantially similar sequence thereof.
In one embodiment of the disclosure, the antibody comprises an Fc region or the antigen-binding fragment is fused to an Fc region, and the antibody or antigen-binding fragment thereof has an N-glycan on the Fc region, and the N-glycan is selected from the group consisting of Man5GlcNAc2 (Man5), GlcNAc2Man3GlcNAc2(Fuc) (G0F), GalGlcNAc2Man3GlcNAc2(Fuc) (G1F), Gal2GlcNAc2Man3GlcNAc2(Fuc) (G2F), Sia2(α2-3)Gal2GlcNAc2Man3GlcNAc2(Fuc) (G2S2F (alpha 2,3 linkage)), Sia2(α2-6)Gal2GlcNAc2Man3GlcNAc2(Fuc) (G2S2F (alpha 2,6 linkage)), GlcNAc2Man3GlcNAc2 (G0), Gal2GlcNAc2Man3GlcNAc2 (G2), Sia2(α2-3)Gal2GlcNAc2Man3GlcNAc2 (G2S2 (alpha 2,3 linkage)), and Sia2(α2-6)Gal2GlcNAc2Man3GlcNAc2 (G2S2 (alpha 2,6 linkage)).
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24, and the N-glycan is selected from the group consisting of Man5, G0F, G1F and G2F.
In one embodiment of the disclosure, the N-glycan is selected from the group consisting of G0F, G2F, G2S2F (alpha 2,3 linkage), G2S2F (alpha 2,6 linkage), G0, G2, G2S2 (alpha 2,3 linkage), and G2S2 (alpha 2,6 linkage), and a plurality of the antibodies or antigen-binding fragment thereof are provided in a population, and more than about 90% of the population has the same N-glycan.
The present disclosure also provides a kit for enrichment of cells comprising an antibody or antigen-binding fragment thereof, wherein the antibody comprises an Fc region or the antigen-binding fragment is fused to an Fc region, and the antibody or antigen-binding fragment thereof is glycoengineered on the Fc region; and a support for immorbilizing the antibody or antigen-binding fragment thereof.
In one aspect, the present disclosure provides a kit for enrichment of cells comprising an antibody or antigen-binding fragment thereof, wherein the antibody comprises an Fc region, or the antigen-binding fragment is fused to an Fc region to form an Fc-fused antigen-binding fragment, and the antibody or Fc-fused antigen-binding fragment thereof is glycoengineered on the Fc region; and a support for immobilizing the antibody or antigen-binding fragment thereof.
In some embodiments, the kit comprises the antibody or antigen-binding fragment thereof as described herein.
In one embodiment of the disclosure, the support is a magnetic bead.
In one embodiment of the disclosure, the kit further comprises an antigen linked to the support.
In a further embodiment, the antigen is SSEA4. In one embodiment, the antigen binds to the antibody or antigen-binding fragment thereof.
In one embodiment of the disclosure, wherein the antibody or antigen-binding fragment is an anti-SSEA4 antibody or SSEA4-binding fragment.
In another aspect, the present disclosure also provides a method for enrichment of cells comprising:
In another aspect, the present disclosure also provides a method for enrichment of cells comprising:
In one embodiment of the disclosure, the method is for enrichment of immune cells.
In one embodiment of the disclosure, the pool of cells is peripheral blood mononuclear cells. In one more preferred embodiment of the disclosure, the peripheral blood mononuclear cells are selected from the group consisting of natural killer cells, natural killer T cells, macrophages, monocytes and B cells.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof is immobilized on a support.
In one embodiment of the disclosure, the antibody has a fragment antigen binding region (Fab region) or Fc-fused antigen-binding fragment has the antigen-binding fragment, and the Fab region or the antigen-binding fragment is immobilized on the support, and the isolated cells bind to the Fc region.
In one embodiment of the disclosure, the Fab region or the antigen-binding fragment binds to SSEA4 linked on the support.
In one embodiment of the disclosure, the support is a magnetic bead.
In one embodiment of the disclosure, the method further comprises:
In one embodiment of the disclosure, the cells that fail to bind to the antibody or Fc-fused antigen-binding fragment are T cells.
In one embodiment of the disclosure, the antibody or antigen-binding fragment is an anti-SSEA4 antibody or SSEA4-binding fragment.
In an embodiment, more than about 90% of the cells that fail to bind to the antibody or Fc-fused antigen-binding fragment are T cells.
In one embodiment of the disclosure, the method comprises the antibody or antigen-binding fragment thereof as described herein.
The present disclosure is described in detail in the following sections. Other characteristics, purposes and advantages of the present disclosure can be found in the detailed description and claims.
In the description that follows, a number of terms are used and the following definitions are provided to facilitate understanding of the claimed subject matter. Terms that are not expressly defined herein are used in accordance with their plain and ordinary meanings.
Unless otherwise specified, “a” or “an” means “one or more.”
As used herein, the term “epitope” refers to the site on the antigen to which an antibody binds.
The term “antibody”, as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., SSEA4). The term “antibody” includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the disclosure, the FRs of the anti-SSEA4 antibody (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.
As used herein, the term “complementarity determining region” (CDR) refers to the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definitions include overlapping or subsets of amino acid residues when compared against each other.
The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to an epitope in SSEA4 or a fragment thereof. In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof or Fc-fused antigen-binding fragment thereof that specifically binds to an epitope in SSEA4 or a fragment thereof.
In one embodiment, the antibody or antigen-binding fragment thereof (anti-SSEA4) comprises complementarity determining regions (CDRs) of a heavy chain variable region and complementarity determining regions of a light chain variable region, wherein the complementarity determining regions of the heavy chain variable region comprises CDRH1, CDRH2 and CDRH3 regions, and the complementarity determining regions of the light chain variable region comprises CDRL1, CDRL2 and CDRL3 regions, and wherein:
Particularly, the antibody or antigen-binding fragment thereof (anti-SSEA4(2)) comprises:
Particularly, the antibody or antigen-binding fragment thereof (anti-SSEA4(3)) comprises:
The sequence listing is shown in Table 1.
I
DGYRGYNMDY
SQGVYWVRQPPGKGL
SALMSRLSISKDNSK
MDYWGQGTSVTVSS
STLRSRVTISKDNSKSQLFL
GYAMDYWGQGTLVTVSS
YADSVKGRFTISRDNSKNT
PGAGYAMDYWGQGTSVTVSS
GGGGSGGGGSGGGGSENVLT
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 13, the CDRH2 region being SEQ ID NO: 8, the CDRH3 region being SEQ ID NO: 9, the CDRL1 region being SEQ ID NO: 10, the CDRL2 region being SEQ ID NO: 20 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 13, the CDRH2 region SEQ ID NO: 15, the CDRH3 region being SEQ ID NO: 9, the CDRL1 region being SEQ ID NO: 10, the CDRL2 region being SEQ ID NO: 11 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 13, the CDRH2 region being SEQ ID NO: 8, the CDRH3 region being SEQ ID NO: 16, the CDRL1 region being SEQ ID NO: 10, the CDRL2 region being SEQ ID NO: 11 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 14, the CDRH2 region being SEQ ID NO: 8, the CDRH3 region being SEQ ID NO: 17, the CDRL1 region being SEQ ID NO: 10, the CDRL2 region being SEQ ID NO: 21 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 14, the CDRH2 region being SEQ ID NO: 8, the CDRH3 region being SEQ ID NO: 18, the CDRL1 region being SEQ ID NO: 10, the CDRL2 region being SEQ ID NO: 11 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 7, the CDRH2 region being SEQ ID NO: 15, the CDRH3 region being SEQ ID NO: 9, the CDRL1 region being SEQ ID NO: 10, the CDRL2 region being SEQ ID NO: 20 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 7, the CDRH2 region being SEQ ID NO: 15, the CDRH3 region being SEQ ID NO: 17, the CDRL1 region being SEQ ID NO: 10, the CDRL2 region being SEQ ID NO: 11 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises the CDRH1 region being SEQ ID NO: 7, the CDRH2 region being SEQ ID NO: 8, the CDRH3 region being SEQ ID NO: 16, the CDRL1 region being SEQ ID NO: 10, the CDRL2 region being SEQ ID NO: 20 and the CDRL3 region being SEQ ID NO: 12.
In one embodiment of the disclosure, the CDRs of a heavy chain and a light chain are listed in Table 2.
I
DGYRGYNMDY (16)
I
DGYRGYNMDY (16)
Bolded and under-lined letters indicate mutagenesis sites.
All the antibodies disclosed in the present disclosure have a significant improvement in affinity to SSEA4 compared to a wild-type antibody.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of
or a substantially similar sequence thereof and a light chain variable region comprising the amino acid sequence of
FQGSGYPLTFGSGTKLEIKR)
or a substantially similar sequence thereof. The under-lined letters indicate CDR regions. Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. Preferably, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof is a single-chain FAT (scFv) molecule comprising the amino acid sequence of SEQ ID NO: 37.
In one embodiment of the disclosure, the antigen-binding fragment is fused to an Fc region having the amino acid sequence of SEQ ID NO: 38.
The antibody according to the disclosure can be full-length (for example, an IgG1 or IgG4 antibody) or may comprise only an antigen-binding portion (for example, an Fab, F(ab′)2 or scFv fragment), and may be modified to affect functionality as needed.
The antibody or antigen-binding fragment thereof according to the disclosure specifically binds to SSEA4. SSEA4 is a hexasaccharide belonged to globo-series glycosphingolipids (GSLs) and comprises the structure of Neu5Acα2→3 Galβ1→3 GalNAcβ1→3 Galα1→4Galβ1→4Glcβ1. SSEA4 is overexpressed in many epithelial cancers, including ovarian, gastric, prostate, lung, breast, and pancreatic cancers, as well as in renal cell carcinoma and glioblastoma multiforme.
The antibody described herein also includes an antigen-binding fragment of a full antibody molecule. An antigen-binding fragment of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
Non-limiting examples of an antigen-binding fragment includes: (i) Fab fragments; (ii) to F(ab′)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment,” as used herein.
An antigen-binding fragment of an antibody typically comprises at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.
In certain embodiments, an antigen-binding fragment may fuse to at least one constant domain, such as an Fc region, namely an Fc-fused antigen-binding fragment. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present disclosure include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3, (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In some embodiments of the disclosure, the antigen-binding fragment is VH-VL, and the Fc-fused antigen-binding fragment is VH-VL-Fc. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains, the adjacent variable domains, or the adjacent constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region.
In some embodiments of the disclosure, the antigen-binding fragment is VH-linker-VL, and the Fc-fused antigen-binding fragment is VH-linker-VL-Fc. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. In some embodiments of the disclosure, the linker region is three copies of G4S (SEQ ID NO: 39). Moreover, an antigen-binding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
As with a full antibody molecule, an antigen-binding fragment may be monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art.
Preferably, the antibody or antigen-binding fragment thereof according to the disclosure is a mammalian antibody.
The term “mammalian antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from mammalian germline immunoglobulin sequences. The mammalian antibodies of the disclosure may include amino acid residues not encoded by mammalian germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
Mammalian antibodies such as human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, an immunoglobulin molecule comprises a stable four chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain heavy chain disulfide bond. In a second form, the dimers are not linked via inter-chain disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently coupled light and heavy chain (half-antibody). These forms have been extremely difficult to separate, even after affinity purification.
The anti-SSEA4 antibody disclosed herein comprises one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The present disclosure includes an antibody, and an antigen-binding fragment thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another mammalian germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR residues within the VH and/or VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and/or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present disclosure.
The present disclosure also includes an anti-SSEA4 antibody comprising variants of any of the VH, VL, and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes an anti-SSEA4 antibody having VH, VL, and/or CDR amino acid sequences with, e.g., 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, etc. conservative amino acid substitutions relative to any of the VH, VL, and/or CDR amino acid sequences disclosed herein.
As applied to polypeptides, the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, to alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutant thereof. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each herein incorporated by reference.
Preferably, the antibody according to the disclosure is a monoclonal antibody.
The antibodies of the present disclosure may be monospecific, bi-specific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. The anti-SSEA4 antibodies of the present disclosure can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bi-specific or a multispecific antibody with a second binding specificity. For example, the present disclosure includes bi-specific antibodies wherein one arm of an immunoglobulin is specific for SSEA4 or a fragment thereof, and the other arm of the immunoglobulin is specific for a second therapeutic target or is conjugated to a therapeutic moiety.
In one embodiment of the disclosure, the antibody or antigen-binding fragment thereof can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is a mammalian cell expression, such as a hybridoma, or a CHO cell expression system. Many such systems are widely available from commercial suppliers. In embodiments in which an antibody comprises both a VH and VL region, the VH and VL regions may be expressed using a single vector, e.g., in a di-cistronic expression unit, or under the control of different promoters. In other embodiments, the VH and VL region may be expressed using separate vectors. A VH or VL region as described herein may optionally comprise a methionine at the N-terminus.
The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby, Immunology (3.sup.rd ed. 1997)).
Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Pat. Nos. 4,946,778, 4,816,567) can be adapted to produce antibodies to polypeptides of this disclosure. Also, transgenic mice, or other organisms such as other mammals, can be used to express humanized or human antibodies (see, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).
Preferably, the antibody or antigen-binding fragment thereof according to the present disclosure is a glycoantibody. The term “glycoantibody” as used herein refers to a homogeneous population of monoclonal antibodies having a single, uniform glycoform on Fc region. The individual glycoantibodies in the homogeneous population are identical, bind to the same epitope, and contain the same Fc glycan with a well-defined glycan structure and sequence.
The term “homogeneous” in the context of a glycosylation profile of Fc region is used interchangeably and are intended to mean a single glycosylation pattern represented by one desired N-glycan species, with little or no trace amount of precursor N-glycan. In certain embodiments, the trace amount of the precursor N-glycan is less than about 2%.
As used herein, the term “glycan” refers to a polysaccharide, oligosaccharide or monosaccharide. Glycans can be monomers or polymers of sugar residues and can be linear or branched. A glycan may include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and/or modified sugars (e.g., 2′-fluororibose, 2′-deoxyribose, phosphomannose, 6′ sulfo N-acetylglucosamine, etc). Glycan is also used herein to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide or a proteoglycan. Glycans usually consist solely of O-glycosidic linkages between monosaccharides. For example, cellulose is a glycan (or more specifically a glucan) composed of β-1,4-linked D-glucose, and chitin is a glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can be homo or heteropolymers of monosaccharide residues, and can be linear or branched. Glycans can be found attached to proteins as in glycoproteins and proteoglycans. They are generally found on the exterior surface of cells. O- and N-linked glycans are very common in eukaryotes but may also be found, although less commonly, in prokaryotes. N-Linked glycans are found attached to the R-group nitrogen (N) of asparagine in the sequon. The sequon is an Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid except praline.
As used herein, the term “N-glycan” refers to an N-linked oligosaccharide attached by an N-acetylglucosamine (GlcNAc) linked to the amide nitrogen of an asparagine residue in an Fc-containing polypeptide.
In one preferred embodiment of the disclosure, the antibody or antigen-binding fragment thereof has a glycol-engineered N-glycan. The antibody comprises an Fc region or the antigen-binding fragment is fused to an Fc region, and the antibody or antigen-binding fragment thereof has an N-glycan on the Fc region, and the N-glycan is selected from the group consisting of Man5GlcNAc2 (Man5), GlcNAc2Man3GlcNAc2(Fuc) (G0F), GalGlcNAc2Man3GlcNAc2(Fuc) (G1F), Gal2GlcNAc2Man3GlcNAc2(Fuc) (G2F), Sia2(α2-3)Gal2GlcNAc2Man3GlcNAc2(Fuc) (G2S2F (alpha 2,3 linkage)), Sia2(α2-6)Gal2GlcNAc2Man3GlcNAc2(Fuc) (G2S2F (alpha 2,6 linkage)), GlcNAc2Man3GlcNAc2 (G0), Gal2GlcNAc2Man3GlcNAc2 (G2), Sia2(α2-3)Gal2GlcNAc2Man3GlcNAc2 (G2S2 (alpha 2,3 linkage)), and Sia2(α2-6)Gal2GlcNAc2Man3GlcNAc2 (G2S2 (alpha 2,6 linkage)).
In one preferred embodiment of the disclosure, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24, and the N-glycan is selected from the group consisting of Man5, G0F, G1F and G2F. Specifically, a plurality of the antibodies or antigen-binding fragment thereof are provided in a population.
The schematic structures of the N-glycan are shown in
In another aspect, the antibody or antigen-binding fragment thereof according to the disclosure has a defucosylated glycoform. As used herein, the term “defucosylated” refers to the absence of a core fucose in the N-glycan of an Fc region. Preferably, the engineered N-glycan is selected from the group consisting of G0, G2, G2S2 (alpha 2,3 linkage), and G2S2 (alpha 2,6 linkage).
In another aspect, the antibody or antigen-binding fragment thereof according to the disclosure has a glycoengineered Fc. As used herein, the term “glycoengineered Fc” when used herein refers to N-glycan on the Fc region has been altered or engineered either enzymatically or chemically. The term “Fc glycoengineering” as used herein refers to the enzymatic or chemical process used to make the glycoengineered Fc. Exemplary methods of engineering are described in, for example, Wong et al U.S. Ser. No. 12/959,351, the contents of which is hereby incorporated by reference. Preferably, the engineered N-glycan is selected from the group consisting of G0F, G2F, G2S2F (alpha 2,3 linkage), G2S2F (alpha 2,6 linkage), G0, G2, G2S2 (alpha 2,3 linkage), and G2S2 (alpha 2,6 linkage), and a plurality of the antibodies or antigen-binding fragment thereof are provided in a population, and more than about 90% of the population has the same N-glycan.
The schematic structures of the engineered N-glycan are shown in Table 4.
In some embodiments, a plurality of the antibodies or antigen-binding fragment thereof are provided in a population, and more than about 90% of the population of the antibody or antigen-binding fragment thereof has the same N-glycan.
In another aspect, the present disclosure provides a method for enrichment of cells comprising:
In another aspect, the present disclosure also provides a method for enrichment of cells comprising:
The present disclosure also provides a kit for enrichment of cells comprising an antibody or antigen-binding fragment thereof, wherein the antibody comprises an Fc region or the antigen-binding fragment is fused to an Fc region, and the antibody or antigen-binding fragment thereof is glycoengineered on the Fc region; and a support for immobilizing the antibody or antigen-binding fragment thereof.
In one aspect, the present disclosure provides a kit for enrichment of cells comprising an antibody or antigen-binding fragment thereof, wherein the antibody comprises an Fc region, or the antigen-binding fragment is fused to an Fc region to form an Fc-fused antigen-binding fragment, and the antibody or Fc-fused antigen-binding fragment thereof is glycoengineered on the Fc region, and a support for immobilizing the antibody or antigen-binding fragment thereof.
In another embodiment of the disclosure, the kit further comprises an antigen. The antigen can link to the support. In some embodiments of the disclosure, the antigen binds to the antibody or antigen-binding fragment thereof. Preferably, the antigen is SSEA4.
In one embodiment of the disclosure, the antibody or antigen-binding fragment is an anti-SSEA4 antibody or SSEA4-binding fragment.
The immune cells according to the disclosure are antibody-binding cells. In one embodiment of the disclosure, the immune cells bind to an Fab region of the antibody or antigen-binding fragment thereof In one another embodiment of the disclosure, the immune cells bind to an Fc region of the antibody or Fc-fused antigen-binding fragment thereof. In some embodiments of the disclosure, the immune cells are selected from the group consisting of natural killer cells, natural killer T cells, macrophages, monocytes and B cells.
As used herein, the term “enrichment” refers to a process of increasing a proportion of the immune cells in a sample. Preferably, the enrichment is a process of partial purification or a partial selection.
As used herein, the term “a pool of cells” refers to a group of cells containing the immune cells. Normally, the pool of cells according to the disclosure comprises other cells. Preferably, the pool of cells is a sample derived from a subject. As used herein, the term “sample” encompasses a variety of sample types obtained from an individual, subject or patient. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. As interchangeably used herein, the terms “individual,” “subject,” and “patient,” refer to a mammal, including, but not limited to, murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc. In one preferred embodiment of the disclosure, the pool of cells is peripheral blood mononuclear cells. More preferably, the peripheral blood mononuclear cells are selected from the group consisting of natural killer cells, natural killer T cells, macrophages, monocytes and B cells
When the method according to the disclosure is applied for the enrichment of the immune cells binding to an Fc region of the antibody or Fc-fused antigen-binding fragment thereof and an Fab region or the antigen-binding fragment is immobilized on a support.
When the method according to the disclosure is applied for the enrichment of the immune cells binding to an Fc region, the antibody or Fc-fused antigen-binding fragment thereof has an Fc region and an Fab region or an antigen-binding fragment, the Fab region or the antigen-binding fragment binds to SSEA4 linked on a support, and the isolated cells bind to the Fc region. The schematic diagram is shown in
Not willing limited by theory, it is believed that the glycoengineering according to the disclosure changes the structure of the Fc region and improves the binding to cells with Fc receptors. Therefore, the capture of cells with Fc receptors is easier and more efficient.
The manners for immobilizing the Fab region on the support or linking the SSEA4 to the support may be direct or indirect chemical bonding. In one preferred embodiment of the disclosure, a biotin-streptavidin reaction may be utilized in such immobilization of linkage.
The support according to the disclosure refers to a solid. Examples of the support include but are not limited to a polymer, a metal or a magnetic solid. In one preferred embodiment of the disclosure, the support is a magnetic bead, which benefits the isolating step. Preferably, the diameter of the magnetic bead is less than about 1 μm.
The manner for isolating the cells binding to the antibody or Fc-fused antigen-binding fragment thereof may be varied according to the property of the support. For example, when the support is a magnetic bead, a magnetic force or centrifuge may be applied for such isolation.
In one embodiment of the disclosure, a substantially pure T cell population can be obtained and enriched. In some embodiments of the disclosure, more than about 90% of the cells that fail to bind to the antibody or Fc-fused antigen-binding fragment are T cells. The method further comprises:
It is believed, though not intended to be restricted by any theoretical, by removing the cells binding to the antibody or Fc-fused antigen-binding fragment thereof, the cells can be further isolated as a substantially pure T cell population. In order to facilitate the exclusion, the antibody or antigen-binding fragment thereof may be immobilized on a support. In one preferred embodiment of the disclosure, the antibody or antigen-binding fragment is an anti-SSEA4 antibody or SSEA4-binding fragment.
In some embodiments, the antibody or antigen-binding fragment thereof used in the method of the present disclosure is any one of the embodiments describe therein.
In some embodiments of the disclosure, peripheral blood mononuclear cells (PBMCs) are first isolated from a whole blood sample derivated from a subject, and an Fc-enriched population is further obtained by the method or kit for enrichment of cells as disclosed herein. The obtained Fc-enriched population may be further subjected to expansion for therapy/storage uses.
The following examples are provided to aid those skilled in the art in practicing the present disclosure.
scFv-SSEA4 (VH-linker-VL) used herein comprises an anti-SSEA4 heavy chain hMC41 (SEQ ID NO: 23) and light chain (SEQ ID NO: 24) and three copies of G4S linker inbetween.
Fc-fused antigen-binding fragment (VH-linker-VL-Fc) comprises an anti-SSEA4 heavy chain hMC41 (SEQ ID NO: 23), light chain (SEQ ID NO: 24), three copies of G4S linker to inbetween and an Fc region (SEQ ID NO: 38).
A series of mutations of anti-SSEA4 antibodies are also constructed.
Briefly, 2×105 cells were incubated with antibody at concentration of 20 nM in FACS buffer (2% FBS in PBS) at 4° C. for 1 hr. After washes with FACS buffer, cells were then incubated with FITC-conjugated goat anti-human IgG antibody (1:250 diluted in FACS buffer, Jackson Immuno Research) for 30 minutes at 4° C. The detection of anti-SSEA4 antibodies bound to cells was analyzed by BD FACSVerse flow cytometer.
As shown in
The antigen binding affinity of anti-SSEA4 antibodies were determined by ELISA. Briefly, antibodies were diluted in PBS at concentration of 0.5 mg/mL and then allowed to incubate with SSEA4 in 96-well assay plates for 2 hours at room temperature. Following the wash cycles, HRP-conjugated goat anti-human IgG antibody (1:10,000 diluted in PBS, Jackson Immuno Research) was added to wells and incubated at room temperature for another one hour. After the wash cycles, TMB ELISA substrate (Thermo scientific) was added for color development, and the reaction was stopped by adding equal volume of 2.5N H2SO4. The absorbance at O.D. 450 nm was read and recorded by M5 ELISA reader (Molecular Device). Position of amino acid changes was presented as wild type amino acid preceding number and mutation following. As shown in in
The method for enrichment of cells was carried out as the following scheme.
The results of flow cytometry are shown in
The results of comparison of antibodies with (anti-SSEA4-G2S2 α2,6) or without (anti-SSEA4) glycoengineering in immune cells capturing are shown in
The results of comparison of antibodies with (anti-SSEA4-G2S2 α2,6) or (anti-SSEA4(2)-G2S2 α2,6) in immune cells capturing are shown in
The results of comparison of antibodies with (anti-SSEA4-G2S2 α2,6) or (anti-SSEA4(3)-G2S2 α2,6) in immune cells capturing are shown in
The results of immune cells enrichment using antibodies with different antigen binding affinities and the individual cell binding intensities are shown in
The results of comparison of antibodies with anti-SSEA4, anti-SSEA4(2) or anti-SSEA4(3) in the individual cell binding intensities are shown in
It can be seen that the ability of cell binding intensities by the Fc region of anti-SSEA4 antibodies having different Fab regions is different. However, referring to
The captured immune cells were cultured with IL-2 and a feeder cells (inactivated K562) with PRIME-XV medium. The results of cell expansion and profile of cultured cells are shown in
The captured immune cells captured by antibodies with (anti-SSEA4-G2S2 α2,6) or without (anti-SSEA4) glycoengineering were cultured with IL-2 and a feeder cells (inactivated K562) with PRIME-XV medium. The results of cytotoxicity on day 14 are shown in
The captured immune cells captured by antibodies with (anti-SSEA4-G2S2 α2,6) or without (anti-SSEA4) glycoengineering and by anti-CD56 antibodies were cultured with IL-2 and a feeder cells (inactivated K562) with PRIME-XV medium. The results of cytotoxicity on day 14 are shown in
The results demonstrate that the enriched cells showed better direct killing activity to K562 target cells than parental PBMC or cells enriched by anti-CD56 beads (a general method for isolation of NK cells).
The conventional approach for T cell isolation was performed by Miltenyi Biotech Pan T Cell Isolation Kit (Order no. 130-596-535). Non-T cells are labeled with a cocktail of biotin-conjugated monoclonal antibodies and the Pan T Cell MicroBeads Cocktail. The magnetically labeled non-T cells are depleted by retaining them within a magnetic column, while the unlabeled T cells flow through the column.
The method of the disclosure was described below. 5×105 T cells per well in 12 well plate were spin-inoculated with CAR lentivirus 24 hrs after addition of CD3/CD28 Dynabeads. The virus were added to activated T cells at MOI=10 with 8 mg/mL polybrene and spin at 800 g, 32° C., for 90 minutes. A few hours later, replenish with IL2 containing fresh medium. 4 days after transduction, remove dynabeads. After culturing for ˜10 days (expand cells and renew IL2 every 2-3 days), the cells were applied for examination of CAR+ percentage and cytotoxic activity.
In contrast to the T cells isolation with conventional approach (Ctrl T) that requires antibodies cocktails to deplete non-T cells, only one antibody is enough here to obtain pure and high quality of CD3+ T cells. As shown in
While the present disclosure has been described in conjunction with the specific embodiments set forth above, many alternatives thereto and modifications and variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are regarded as falling within the scope of the present disclosure.
This application claims priority from U.S. 63/119,345 filed 30 Nov. 2020, the contents of which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63119345 | Nov 2020 | US |
