Patent Application
20240156870
The present disclosure relates generally to anti-EGFR antibodies. More particularly, the present disclosure relates to ant-EGFR single domain antibodies.
Cancer is a major public health problem and the second leading cause of death worldwide. Traditional therapy for cancer has included surgery, radiation and chemotherapy. These have been moderately successful for treatment of some cancers, particularly those diagnosed at early stages. However effective therapy is lacking for many aggressive cancers. Recent technological innovations suggest that immunotherapy (stimulating or restoring a patient's own immune system to fight cancer) can potentially provide potent and long term responses against many cancers including aggressive hard to treat cancers.
Immunotherapy has had phenomenal success in treating certain cancers. Antibodies have been used alone and in therapeutic constructs.
For example, bi-specific T cell engagers, and bi- and tri-specific killer cell engagers (BiKEs and TriKEs) incorporating single-chain variable fragments (scFvs) have been developed to direct a host's immune system to target cancer cell.
Chimeric Antigen Receptor (CAR) constructs have also been produced to combined facets of T cell activation into a single protein. These molecules link an extracellular antigen recognition domain to an intracellular signaling domain, which activates the T cell when an antigen is bound. Chimeric Antigen Receptor (CAR) modified immune cell therapies are an emergent form of cancer immunotherapy whereby single or multiple antigen binding domains from antibodies that specifically target cell surface protein(s) on cancer cells are combined with immune cell activating domains to generate “armored” Immune cells that seek and kill specific cells that harbor the targeting antigen(s). CAR modified T cell therapies (CAR-T) have provided unprecedented responses for patients suffering from certain aggressive forms of leukemia, for example.
Up-regulation of epidermal growth factor receptor (EGFR) is a hallmark of many solid tumors, and inhibition of EGFR signaling by small molecules and antibodies has clear clinical benefit. EGFR is a receptor tyrosine kinase that is overexpressed and constitutively activated in up to 80% of solid cancers [22]. Following the development of small molecule inhibitors, naked antibodies (Abs) against EGFR, exemplified by cetuximab, have shown clinical benefit in treating colorectal [23] and head and neck cancers [24]. Other EGFR Abs are under investigation in other indications and as Ab-drug conjugates. Camelid single-domain antibodies (sdAbs or VHHs) have previously been isolated against EGFR using protein immunization [25] and whole-cell immunization [26], and biparatopic molecules with improved potency have been constructed from these sdAb building blocks [27]. One advantage of sdAb-based biologics for cancer therapy is that they tend to penetrate solid tumors better than full-length IgGs [28].
It is, therefore, desirable to provide immunogenic molecule with affinity for EGFR.
It is an object of the present disclosure to obviate or mitigate at least one disadvantage of previous
In a first aspect, there is provided an isolated single domain antibody (sdAb), which binds specifically to human EGFR, the sdAb comprising:
In another aspect, there is provided an isolated single domain antibody (sdAb), which binds specifically to human EGFR, the sdAb comprising:
In another aspect, there is provided an isolated single domain antibody (sdAb), which binds specifically to human EGFR, the sdAb comprising:
In another aspect, there is provided an isolated VHH single domain antibody (sdAb), which binds specifically to human EGFR, the sdAb comprising:
In one aspect, there is provided a recombinant polypeptide comprising an sdAb as defined herein.
In aspect, there is provided a recombinant nucleic acid molecule encoding an sdAb, the recombinant polypeptide, or the VHH:Fc fusion as defined herein.
In one aspect, there is provided a composition comprising an sdAb as defined herein, or a polypeptide comprising such an sdAb; together with an acceptable excipient, diluent or carrier.
In one aspect, there is provided a use of the sdAb as defined herein or of an antibody comprising one or more VHH:Fc fusion as defined herein for treatment of a cancer.
In one aspect, there is provided a use of the sdAb as defined herein or of an antibody comprising one or more VHH:Fc fusion as defined herein for preparation of a medicament for treatment of a cancer.
In one aspect, there is provided the sdAb as defined herein or of an antibody comprising one or more VHH:Fc fusion as defined herein for use in treatment of a cancer.
In one aspect, there is provided a method of treating a cancer in subject comprising administering to the subject the sdAb as defined herein or of an antibody comprising one or more VHH:Fc fusion as defined herein.
In one aspect, there is provided a multivalent antibody comprising an sdAb as defined above.
In aspect, there is provided a recombinant nucleic acid molecule encoding the multivalent antibody as defined herein.
In one aspect, there is provided a composition comprising a multivalent antibody as defined herein; together with an acceptable excipient, diluent or carrier.
In one aspect, there is provided a use of the multivalent antibody as defined herein for treatment of a cancer.
In one aspect, there is provided a use of the multivalent antibody as defined herein for preparation of a medicament for treatment of a cancer.
In one aspect, there is provided the multivalent antibody as defined herein for use in treatment of a cancer.
In one aspect, there is provided a method of treating a cancer in subject comprising administering to the subject the multivalent antibody as defined herein.
In one aspect, there is provided a chimeric antibody receptor (CAR), which binds to human EGFR, comprising the VHH sdAb as defined herein.
In one aspect, there is provided a recombinant nucleic acid molecule encoding the CAR as defined herein.
In one aspect, there is provided a vector comprising the recombinant nucleic acid molecule as defined herein.
In one aspect, there is provided a recombinant viral particle comprising the recombinant nucleic acid as defined herein.
In one aspect, there is provided a cell comprising the recombinant nucleic acid molecule as defined herein.
In one aspect, there is providing a use of the nucleic acid, vector, or viral particle as described herein for preparation of cells for CAR-T.
In one aspect, there is providing a method of preparing cells for CAR-T comprising contacting a T-cell with the viral particle as described herein. In one embodiment, the T-cell is from a donor. In one embodiment, the T-cell is from a patient.
In one aspect, there is providing a method of preparing cells for CAR-T comprising introducing into a T-cell the nucleic acid or vector as described herein. In one embodiment, the T-cell is from a donor. In one embodiment, the T-cell is from a patient.
In one aspect, there is provided a use of the CAR or of the engineered cell as described herein for treatment of a cancer.
In one aspect, there is provided a use of the CAR or of the engineered cell as described herein for preparation of a medicament treatment of a cancer.
In one aspect, there is provided the CAR or the engineered cell as described herein for use in treatment of a cancer.
In one aspect there is provided a method of treating a cancer in a subject, comprising administering to the subject the engineered cell as defined herein.
Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures.
Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.
Generally, the present disclosure provides anti-EGFR single domain antibodies (sdAb) prepared by immunizing a llama with recombinant human EGFRvIII. VHH antibodies specific to EGFR were isolated. The example antibodies initially produced comprise CDR1, CDR2, and CDR3 sequences corresponding, respectively to SEQ NOs: 1-3, 4-6, 7-9, 10-12, 13-15, 16-18, 19-21, 22-24, 25-27, 28-30, 31-33, 34-36, 37-39, 40-42, 43-45, and 46-48; and related sequences, including humanized variants. Also provided are multivalent antibodies comprising any one of the sdAbs, including bispecific T-cell engagers, bispecific killer cell engagers (BiKEs), and trispecific killer cell engagers (TriKEs). Also described are chimeric antigen receptors (CARs) for CAR-T therapy comprising any one of the aforementioned sdAbs. Uses of these molecules in the treatment of cancer are also described, in particularly EGFR-high cancers. Hinge lengths may be selected to achieve desired activities, such as high activity or high selectivity for target vs. non-target cells.
Single Domain Antibodies & Polypeptides Comprising Them
A single domain antibody (sdAb), also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. sdAbs have been derived from heavy-chain antibodies found in Camelidae species (such as camel, llama, dromedary, alpaca and guanaco) using molecular biology techniques, which are also known as VHH fragments (herein also termed “VHH” or “VHH”). Other examples include VNARfragments derived from heavy chain antibodies found in cartilaginous fish, such as sharks. sdAbs have also been generated from a heavy chain/light chain of conventional immunoglobulin G (IgGs) by engineering techniques.
VHH molecules are about 10 times smaller than IgG molecules. These single polypeptides are generally quite stable, often resisting extreme pH and temperature conditions that can be problematic for conventional antibodies and antibody fragments. Moreover, VHHs tend to be more resistant to the action of proteases. Furthermore, in vitro expression of VHHs tends to produce high yield of properly folded/functional VHHs. In addition, heavy chain antibodies and their engineered fragments (i.e., VHHs) generated in Camelidae species may recognize cryptic or hidden epitopes which otherwise inaccessible to larger conventional antibodies and antibody fragments generated in vitro through the use of antibody libraries or by immunization of other mammals.
In one aspect, there is provided an isolated single domain antibody (sdAb), which binds specifically to human EGFR, the sdAb comprising:
In the above, group a) provides consensus sequences defined by antibodies herein termed sdAb021, sdAb022, sdAb023, sdAb024, sdAb025, sdAb026, and sdAb027.
“CDRs” or “complementarity-determining regions” are the portion of the variable chains in immunoglobulins that collectively constitute the paratope, and thereby impart binding specificity and affinity to the antibody. As used here, the term refers to CDRs mapped in sdAbs according to the standards or conventions set by IMGT™ (international ImMunoGeneTics information system).
The antibodies described herein have been raised to the human soluble EGFRvIII (e.g., see UniProt P00533: residues 1-29/Gly/297-645) or membrane-tethered human EGFRvII (e.g., see UniProt P00533: residues 1-29/Gly/297-668) expressed via DNA immunization of llama.
In another aspect, there is provided an isolated single domain antibody (sdAb), which binds specifically to human EGFR, the sdAb comprising:
In one embodiment, the antibody comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 amino acid sequence as set forth in SEQ ID NO: 2, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 3 (from NRC-sdAb02l).
In one embodiment, the antibody comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 4, a CDR2 amino acid sequence as set forth in SEQ ID NO: 5, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 6 (from NRC-sdAb02l).
In one embodiment, the antibody comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 7, a CDR2 amino acid sequence as set forth in SEQ ID NO: 8, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 9 (from NRC-sdAb023).
In one embodiment, the antibody comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 10, a CDR2 amino acid sequence as set forth in SEQ ID NO: 11, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 12 (from NRC-sdAb024).
In one embodiment, the antibody comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 13, a CDR2 amino acid sequence as set forth in SEQ ID NO: 14, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 15 (from NRC-sdAb025).
In one embodiment, the antibody comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 16, a CDR2 amino acid sequence as set forth in SEQ ID NO: 17, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 18 (from NRC-sdAb026).
In one embodiment, the antibody comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 amino acid sequence as set forth in SEQ ID NO: 20, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 21 (from NRC-sdAb027)
In one embodiment, the antibody comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 22, a CDR2 amino acid sequence as set forth in SEQ ID NO: 23, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 24 (from NRC-sdAb028).
In one embodiment, the antibody comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 25, a CDR2 amino acid sequence as set forth in SEQ ID NO: 26, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 27 (from NRC-sdAb032).
In one embodiment, the antibody comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 28, a CDR2 amino acid sequence as set forth in SEQ ID NO: 29, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 30 (from NRC-sdAb033).
In one embodiment, the antibody comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 31, a CDR2 amino acid sequence as set forth in SEQ ID NO: 32, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 33 (from NRC-VH54).
In another aspect, there is provided an isolated single domain antibody (sdAb), which binds specifically to human EGFR, the sdAb comprising:
In one embodiment, in B) the CDR1 CDR2, and CDR3 amino acid sequences are at least 90% identical to the CDR1, CDR2, and CDR3 sequences defined in any one of part A) i) to xxviii). In one embodiment, in B) the CDR1 CDR2, and CDR3 amino acid sequences are at least 95% identical to the CDR1, CDR2, and CDR3 sequences defined in any one of part A) i) to xxviii). In one embodiment, in B) the CDR1 CDR2, and CDR3 amino acid sequences have at most three substitutions compared to the CDR1, CDR2, and CDR3 sequences defined in any one of part A) i) to xxviii). In one embodiment, in B) the CDR1 CDR2, and CDR3 amino acid sequences have at most two substitutions compared to the CDR1, CDR2, and CDR3 sequences defined in any one of part A) i) to xxviii). In one embodiment, in B) the CDR1 CDR2, and CDR3 amino acid sequences have at most one substitution compared to the CDR1, CDR2, and CDR3 sequences defined in any one of part A) i) to xxviii). In some embodiment, sequence differences vs. the sequences set forth in A) are conservative sequence substitutions.
The term “conservative amino acid substitutions” which is known in the art is defined herein as follows, with conservative substitutable candidate amino acids showing in parentheses: Ala (Gly, Ser); Arg (Gly, Gln); Asn (Gln; His); Asp (Glu); Cys (Ser); Gln (Asn, Lys); Glu (Asp); Gly (Ala, Pro); His (Asn; Gln); Ile (Leu; Val); Leu (lie; Val); Lys (Arg; Gln); Met (Leu, lie); Phe (Met, Leu, Tyr); Ser (Thr; Gly); Thr (Ser; Val); Trp (Tyr); Tyr (Trp; Phe); Val (lie; Leu).
Sequence variants according to certain embodiments are intended to encompass molecules in which binding affinity and/or specificity is substantially unaltered vs. the parent molecule from which it is derived. Such parameters can be readily tested, e.g., using techniques described herein and techniques known in the art. Such embodiments may encompass sequence substitutions, insertions, or deletions.
In another aspect, there is provided an isolated VHH single domain antibody (sdAb), which binds specifically to human EGFR, the sdAb comprising:
Recognizing that CDR3 is often the major determinant of binding for VHH sdAbs, it would be understood that other CDRs could be mutagenized or otherwise diversified and a resulting library (or candidate molecule) screened for antibodies that bind to human EGFR and/or cross-compete for binding to EGFR with the parent molecule. These embodiments are intended to cover, inter alia, molecules identified in this manner.
In one embodiment, the isolated sdAb comprises:
In some embodiments, the sdAb cross-reacts with mouse EGFR.
In some embodiments, the sdAb cross-reacts with rhesus EGFR.
These embodiments are intended to encompass, inter alia, embodiments in which molecules recovered following mutagenization/diversification and screening for variant molecules that bind to EGFR and/or cross-compete for binding to EGFR with the parent molecule from which they are derived. As above, a library could be screened or individual candidate molecules could be tested.
In one embodiment, sdAb comprises A) the amino acid sequence of any one of SEQ ID NO: 34 to 48 across the full length thereof, or B) an amino acid sequence that is at least 80% identical to any one of SEQ ID NO: 34 to 48 across the full length thereof. In one embodiment, the amino acid sequence of B) is at least 85% identical across the full length therefore to one of the amino acid sequences of A). In one embodiment, the amino acid sequence of B) is at least 90% identical across the full length therefore to one of the amino acid sequences of A). In one embodiment, the amino acid sequence of B) is at least 95% identical across the full length therefore to one of the amino acid sequences of A). In one embodiment, the amino acid sequence of B) is at least 98% identical across the full length therefore to one of the amino acid sequences of A). In one embodiment, the amino acid sequence of B) is at least 98% identical across the full length therefore to one of the amino acid sequences of A). In some of these embodiments, sequences differences vs. sequences of A) are outside the CDR sequences.
In one embodiment, the sdAb comprises A) the amino acid sequence of any one of SEQ ID NO: 34 to 48.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 34.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 35.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 36.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 37.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 38.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 39.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 40.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 41.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 42.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 43.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 44.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 45.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 46.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 47.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 48.
In one embodiment of the above, CDR1, CDR2, and CDR3 are defined with respect to the IMGT numbering system. It is to be appreciated that CDR sequences could be defined by other conventions, such as the Kabat, Chothia, or EU numbering systems.
In one embodiment, the sdAb comprises SEQ ID NO: 34.
In one embodiment, the sdAb comprises SEQ ID NO: 35.
In one embodiment, the sdAb comprises SEQ ID NO: 36.
In one embodiment, the sdAb comprises SEQ ID NO: 37.
In one embodiment, the sdAb comprises SEQ ID NO: 38.
In one embodiment, the sdAb comprises SEQ ID NO: 39.
In one embodiment, the sdAb comprises SEQ ID NO: 40.
In one embodiment, the sdAb comprises SEQ ID NO: 41.
In one embodiment, the sdAb comprises SEQ ID NO: 42.
In one embodiment, the sdAb comprises SEQ ID NO: 43.
In one embodiment, the sdAb comprises SEQ ID NO: 44.
In one embodiment, the sdAb comprises SEQ ID NO: 45.
In one embodiment, the sdAb comprises SEQ ID NO: 46.
In one embodiment, the sdAb comprises SEQ ID NO: 47.
In one embodiment, the sdAb comprises SEQ ID NO: 48.
In one embodiment, the sdAb is a Camelidae VHH sdAb.
In one embodiment, the sdAb is a llama VHH sdAb
In one embodiment, the sdAb is humanized Camelidae VHH.
By the term “humanized” as used herein is meant mutated so that immunogenicity upon administration in human patients is minor or nonexistent. Humanizing a polypeptide, according to the present invention, comprises a step of replacing one or more of the Camelidae amino acids by their human counterpart as found in the human consensus sequence, without that polypeptide losing its typical character, i.e. the humanization does not significantly affect the antigen binding capacity of the resulting polypeptide. A humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting, veneering or resurfacing, chain shuffling, etc.
In one embodiment, the humanized antibody comprises SEQ ID NO: 42 (NRC-sdAb028-H1).
In one embodiment, the humanized antibody comprises SEQ ID NO: 44 (NRC-sdAb032-H1).
In one embodiment, the humanized antibody comprises SEQ ID NO: 46 (NRC-sdAb033-H1).
In one embodiment, the humanized antibody comprises SEQ ID NO: 47 (NRC-sdAb033-H2).
In one embodiment, the sdAb has an affinity for human EGFR of 40 nM or less. In one embodiment, the sdAb has an affinity for human EGFR of 20 nM or less. In one embodiment, the sdAb has an affinity for human EGFR of 10 nM or less. In one embodiment, the sdAb has an affinity for human EGFR of 5 nM or less. Binding affinity can be determined, e.g., according to assays described herein.
In one aspect, there is provided a VHH single domain antibody (sdAb) that competes for specific binding to human EGFR with the isolated sdAb described above. An sdAb of the invention may be identified by a method that comprises a binding assay which assesses whether or not a test antibody is able to cross-compete with a known antibody of the invention for a binding site on the target molecule. For example, the antibodies described hereinabove may be used as reference antibodies. Methods for carrying out competitive binding assays are well known in the art. For example they may involve contacting together a known antibody of the invention and a target molecule under conditions under which the antibody can bind to the target molecule. The antibody/target complex may then be contacted with a test antibody and the extent to which the test antibody is able to displace the antibody of the invention from antibody/target complexes may be assessed. An alternative method may involve contacting a test antibody with a target molecule under conditions that allow for antibody binding, then adding an antibody of the invention that is capable of binding that target molecule and assessing the extent to which the antibody of the invention is able to displace the test antibody from antibody/target complexes. Such antibodies may be identified by generating new sdAbs to EGFR and screening the resulting library for cross-competition. Alternatively, one of the antibodies described herein may serve as a starting point for diversification, library generation, and screening. A further alternative could involve testing individual variants of an antibody described herein.
In one embodiment, the sdAb defined herein is a camelid sdAb.
In one embodiment, the sdAb defined herein is a llama sdAb.
In one embodiment, the sdAb defined herein is humanized form of Camelidae sdAb.
Table 1 and Table 7 lists the full length sequences for various sdAb disclosed herein. CDR1, CDR2, and CDR3 sequences are underlined in Table 7. CDR identification and numbering used herein is according to the IMGT™ convention.
Recombinant Polypeptides
In one aspect, there is provided a recombinant polypeptide comprising an sdAb as defined herein. In one embodiment, there is provided a recombinant polypeptide comprising one or more sdAb as defined herein. In one embodiment, there is provided a recombinant polypeptide comprising two or more sdAb as defined herein. In one embodiment, there is provided a recombinant polypeptide comprising two or more sdAb as defined herein.
VHH:Fc Fusions
In one embodiment, there is provided the sdAb defined herein fused to a human Fc (termed a “VHH:Fc fusion”). For example, the VHH:Fc fusion may comprise at least a CH2 and a CH3 of the IgG, IgA, or IgD isotype. The VHH:Fc fusion may comprise at least a CH2, a CH3, and a CH4 of the IgM or IgE isotype. Such embodiments may be useful in activating the immune system in higher order recombinant molecules. For example, according to some embodiments, two such F-containing VHH:Fc fusions may assemble to form a recombinant monomeric antibody. In some embodiment, such a monomeric antibody is capable of activating the immune system. Such monomeric antibodies may be of IgG, IgA, IgD, IgE, or IgM isotype. In one embodiment, IgA Fc-containing VHH:Fc fusions may also assemble into a recombinant dimeric (secretory) form. Multimeric forms are also envisaged in some embodiments. For example, five IgM monomers may assemble to form a recombinant pentameric antibody.
In some embodiments, the multivalent antibody described herein may be an assembly of the same VHH:Fc fusions.
In some embodiments, the multivalent antibody described herein may be an assembly of the different VHH:Fc fusions having the same binding target. For example, these may bind to different epitopes on the same target molecule. Examples may include assemblies of different VHH:Fc fusions, each comprising a different anti-EGFR sdAb as defined herein.
In some embodiments, the multivalent antibody described herein may be an assembly of an VHH:Fc fusion defined herein (comprising an anti-EGFR sdAb as defined herein) and another VHH:Fc fusion comprising a paratope directed to a different target.
Fusions to Cargo Molecules
In a further embodiment, the present disclosure provides an anti-EGFR sdAb as defined herein linked to a cargo molecule. The cargo molecule may comprise, for example, a therapeutic moiety, such as for example, a cytotoxic agent, a cytostatic agent, an anti-cancer agent or a radiotherapeutic. In particular embodiments of the disclosure, the antibody drug conjugates may comprise a cytotoxic agent. Another particular embodiment of the disclosure relates to antibody drug conjugates comprising a radiotherapeutic.
Targeted Nanoparticles
In another aspect, there is provided a nanoparticle linked to an EGFR sdAb as defined herein. The nanoparticle may comprise a therapeutic payload, such as a small molecule, polypeptide, or polynucleotide. The polynucleotide may comprise, for example, an mRNA, an siRNA, or an shRNA. The mRNA may encode a therapeutic polypeptide, such as a cytotoxic molecule. Such nanoparticles may be useful for targeted delivery of the therapeutic payload.
In one embodiment, there is provided a method of delivering a therapeutic payload to a cell, the method comprising contacting the cell with the nanoparticle linked to the EGFR sdAb. The cell may a cancer cell. The method may be a method of treating cancer in a subject.
In another embodiment, there is provided a use of the nanoparticle linked to the EGFR sdAb for delivery of a therapeutic payload to a cell. In another embodiment, there is the nanoparticle linked to the EGFR sdAb for use in delivery of a therapeutic payload to a cell. The cell may a cancer cell. The use may be for treatment of cancer in a subject.
Recombinant Nucleic Acid Molecules
In one aspect, there is provided a recombinant nucleic acid molecule encoding an sdAb, the recombinant polypeptide, or the VHH:Fc fusion as defined herein. In one embodiment, the nucleic acid is DNA. In one embodiment, the nucleic acid is RNA. In one embodiment, the RNA is an mRNA. In one embodiment, the mRNA encodes for a therapeutic.
Compositions
In one aspect, there is provided a composition comprising an sdAb as defined herein, or a polypeptide comprising such an sdAb; together with an acceptable excipient, diluent or carrier. In one embodiment the composition is a pharmaceutical composition, and the excipient, diluent or carrier is a pharmaceutically acceptable excipient, diluent or carrier.
Uses & Methods
In one aspect, there is provided a use of the sdAb as defined herein or of an antibody comprising one or more VHH:Fc fusion as defined herein for treatment of a cancer.
In one aspect, there is provided a use of the sdAb as defined herein or of an antibody comprising one or more VHH:Fc fusion as defined herein for preparation of a medicament for treatment of a cancer.
In one aspect, there is provided the sdAb as defined herein or of an antibody comprising one or more VHH:Fc fusion as defined herein for use in treatment of a cancer.
In one aspect, there is provided a method of treating a cancer in subject comprising administering to the subject the sdAb as defined herein or of an antibody comprising one or more VHH:Fc fusion as defined herein.
In one embodiment, the cancer expresses EGFR. In one embodiment, the cancer aberrantly expresses EGFR normally absent in corresponding healthy tissue of origin and/or surrounding tissue. In one embodiment, the cancer expresses high levels of EGFR compared to corresponding healthy tissue of origin and/or surrounding tissue. In one embodiment, the cancer is selected from the group consisting of (but not limited to) lung cancer, pancreatic cancer, gastric cancer, brain cancer (such as glioblastoma), neuroendocrine cancer, head and neck cancer, bladder cancer, breast cancer, colorectal cancer, cervical cancer, and endometrial cancer.
Multivalent Antibodies & Related Embodiments
In one aspect, there is provided a multivalent antibody comprising an sdAb as defined above.
By “multivalent antibody” is use herein to mean a molecule comprising more than one variable region or paratope for binding to one or more antigen(s) within the same or different target molecule(s).
In some embodiments, the paratopes may bind to different epitopes on the same target molecule. In some embodiments, the paratopes may bind to different target molecules. In these embodiments, the multivalent antibody may be termed bispecific, trispecific, or multispecific, depending on the number of paratopes of different specificity that are present. As the multivalent antibody comprises one of the anti-EGFR sdAbs as herein defined, the multivalent antibody comprises EGFR binding affinity.
For example, as explained above, in some embodiments a multivalent antibody may be an assembly of a VHH:Fc fusion defined herein (comprising an sdAb as defined herein) and another VHH:Fc fusion comprising a different paratope conferring a different specificity.
In one embodiment, there is provided a bispecific antibody comprising an sdAb as defined above, and a second antigen-binding portion. In some embodiments, the second antigen binding portion may comprise a monoclonal antibody, an Fab, and F(ab′)2, an Fab′, an scFv, or an sdAb, such as a VHH or a VNAR.
An “antigen-binding portion” is meant a polypeptide that comprises an antibody or antigen-binding fragment thereof having antigen-binding activity, including engineered antibodies fragments thereof.
In some embodiments, the second antigen-binding portion may bind to human serum albumin, e.g., for the purposes of stabilization/half-life extension.
In one embodiment, there is provided a trispecific antibody comprising an sdAb as defined above, and a second-binding portion, and a third antigen-binding portion. In some embodiments, the second antigen binding portion comprises a monoclonal antibody, an Fab, and F(ab′)2, and Fab′, an sdFv, or an sdAb, such as a VHH or a VNAR. In some embodiments, the third antigen binding portion comprises, independently, a monoclonal antibody, an Fab, and F(ab′)2, and Fab′, an sdFv, or an sdAb, such as a VHH or a VNAR.
The second and/or third antigen-binding portion may bind to human serum albumin, e.g., for the purposes of stabilization/half-life extension.
In some embodiments, the trispecific antibody may be multispecific and the antibody may comprise one or more additional antigen-binding portion(s). In such embodiments, the additional antigen-binding portion(s) may be, independently, an Fab, and F(ab′)2, and Fab′, an sdFv, or an sdAb, such as a VHH or a VNAR.
In one embodiment, the multispecific antibody comprises a first antigen-binding portion comprising an sdAb as defined herein, and a second antigen-binding portion. In one embodiment, the second antigen-binding moiety binds specifically to a cell-surface marker of an immune cell.
A “cell surface marker” is a molecule expressed at the surface of the cell that is particular to (or enriched in) a cell type, and that is capable of being bound or recognized by an antigen-binding portion.
Bispecific T-Cell Engager
In one embodiment, the multivalent antibody is a bispecific T-cell engager comprising an sdAb as defined herein and second antigen-binding moiety that binds specifically to a cell-surface marker of a T-cell. In one embodiment, the T-cell marker comprises human CD3.
Human CD3 is a multi-subunit antigen, of which various subunits may participate in CD3 activation. One such subunit is CD3 epsilon (see, e.g., GenBank NP_000724.1). Other non-limiting examples include CD3 gamma (see, e.g., GenBank NP_000064.1) and delta (see, e.g., GenBank NP_000723.1 for delta isoform A, and, e.g., GenBank NP_001035741.1 for delta isoform B).
In some embodiments, T-cell marker comprises CD3 epsilon, CD3 gamma, or CD3 delta. In one specific embodiment, the T-cell marker comprises CD3 epsilon.
The term “bispecific T-cell engager”, as used herein, refers to a recombinant bispecific protein that has two linked variable regions from two different antibodies, one targeting a cell-surface molecule on T cells (for example, CD3ε), and the other targeting antigens on the surface of disease cells, typically malignant cells. For example a bispecific T-cell engager may comprises an sdAb as defined herein and an scFvs. A bispecific T-cell engager may comprise an sdAb as defined herein and a second VHH/sdAb. The two variable regions are typically linked together by a short flexible linker such as GlySer linker. By binding to tumor antigens and T cells simultaneously, bispecific T-cell engagers mediate T-cell responses and killing of tumor cells. The T-cell/target cell adherence facilitated by a bispecific T-cell engager is independent of MHC haplotype.
In one embodiment, the bispecific T-cell engager comprises in N-terminal to C-terminal direction:
In one embodiment, the signal peptide further comprises a signal peptide N-terminal to the fist antigen-binding portion.
A “signal peptide”, as referred to herein allows the nascent protein to be directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed. The core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases. The signal peptide may be at the amino terminus of the molecule.
In one embodiment, the signal peptide is a signal peptide from human CD28. In one embodiment, the signal peptide from human CD28 comprises SEQ ID NO: 53. In one embodiment, the signal peptide is at least 80% identical to SEQ ID NO: 53. In one embodiment, the signal peptide is at least 90% identical to SEQ ID NO: 53. In one embodiment, the signal peptide is at least 95% identical to SEQ ID NO: 53. In one embodiment, the signal peptide is at least 98% identical to SEQ ID NO: 53.
By “amino acid linker”, in this context, will be understood a sequence of sufficient length, flexibility, and composition to permit the bispecific T-cell engager to be properly functional an engage with both targets.
The amino acid linker may comprise a hinge. The hinge may be from human CD8, e.g. as set forth in SEQ ID NO: 55. In one embodiment, the CD8 hinge domain is at least 80% identical to SEQ ID NO: 55. In one embodiment, hinge domain is at least 90% identical to SEQ ID NO: 55. In one embodiment, the hinge domain is at least 95% identical to SEQ ID NO: 55. In one embodiment, the hinge domain is at least 98% identical to SEQ ID NO: 55.
The hinge may comprise a truncation of SEQ ID NO: 55. The hinge may comprise the C-terminal 22 amino acids of SEQ ID NO: 55 (SEQ ID NO: 57). The hinge may comprise the C-terminal 23 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 24 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 25 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 26 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 27 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 28 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 29 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 30 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 31 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 32 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 33 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 34 amino acids of SEQ ID NO: 55 (SEQ ID NO: 56). The hinge may comprise the C-terminal 35 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 36 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 37 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 38 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 39 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 40 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 41 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 42 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 43 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 44 amino acids of SEQ ID NO: 55. In one embodiment, the hinge comprises a sequence that is at least 80% identical to one of the aforementioned truncations. In one embodiment, the hinge comprises a sequence that is at least 90% identical to one of the aforementioned truncations. In one embodiment, the hinge comprises a sequence that is at least 95% identical to one of the aforementioned truncations. In one embodiment, the hinge comprises a sequence that is at least 98% identical to one of the aforementioned truncations.
In one embodiment, the amino acid linker comprises (in N- to C-terminal direction) SEQ ID NO: 54-SEQ ID NO: 55-SEQ ID NO: 62; or sequences at least 80% identical to SEQ ID NO: 54-SEQ ID NO: 55-SEQ ID NO: 62. In one embodiment, the amino acid linker comprises a sequence that is at least 80% identical to SEQ ID NO: 54. In one embodiment, the amino acid linker comprises a sequence that is at least 90% identical to SEQ ID NO: 54. In one embodiment, the amino acid linker comprises a sequence that is at least 95% identical to SEQ ID NO: 54. In one embodiment, the amino acid linker comprises a sequence that is at least 98% identical to SEQ ID NO: 54. In one embodiment, amino acid linker comprises a sequence that is at least 80% identical to SEQ ID NO: 55. In one embodiment, amino acid linker comprises a sequence that is at least 90% identical to SEQ ID NO: 55. In one embodiment, the amino acid linker comprises a sequence that is at least 95% identical to SEQ ID NO: 55. In one embodiment, the amino acid linker comprises a sequence that is at least 98% identical to SEQ ID NO: 55. In one embodiment, the amino acid linker comprises a sequence that is at least 80% identical to SEQ ID NO: 62. In one embodiment, the amino acid linker comprises a sequence that is at least 90% identical to SEQ ID NO: 62. In one embodiment, the amino acid linker comprises a sequence that is at least 95% identical to SEQ ID NO: 62. In one embodiment, the amino acid linker comprises a sequence that is at least 98% identical to SEQ ID NO: 62.
In one embodiment, the multivalent antibody is encoded by SEQ ID NO: 52.
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 amino acid sequence as set forth in SEQ ID NO: 2, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 3 (from NRC-sdAb021).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 4, a CDR2 amino acid sequence as set forth in SEQ ID NO: 5, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 6 (from NRC-sdAb022).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 7, a CDR2 amino acid sequence as set forth in SEQ ID NO: 8, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 9 (from NRC-sdAb023).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 10, a CDR2 amino acid sequence as set forth in SEQ ID NO: 11, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 12 (from NRC-sdAb024).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 13, a CDR2 amino acid sequence as set forth in SEQ ID NO: 14, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 15 (from NRC-sdAb025).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 16, a CDR2 amino acid sequence as set forth in SEQ ID NO: 17, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 18 (from NRC-sdAb026).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 amino acid sequence as set forth in SEQ ID NO: 20, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 21 (NRC-sdAb027)
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 22, a CDR2 amino acid sequence as set forth in SEQ ID NO: 23, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 24 (from NRC-sdAb028).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 25, a CDR2 amino acid sequence as set forth in SEQ ID NO: 26, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 27 (from NRC-sdAb032).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 28, a CDR2 amino acid sequence as set forth in SEQ ID NO: 29, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 30 (from NRC-sdAb033).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 31, a CDR2 amino acid sequence as set forth in SEQ ID NO: 32, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 33 (from NRC-VH54).
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 34.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 35.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 36.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 37.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 38.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 39.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 40.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 41.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 42.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 43.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 44.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 45.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 46.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 47.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 48.
In one embodiment, the sdAb comprises SEQ ID NO: 34.
In one embodiment, the sdAb comprises SEQ ID NO: 35.
In one embodiment, the sdAb comprises SEQ ID NO: 36.
In one embodiment, the sdAb comprises SEQ ID NO: 37.
In one embodiment, the sdAb comprises SEQ ID NO: 38.
In one embodiment, the sdAb comprises SEQ ID NO: 39.
In one embodiment, the sdAb comprises SEQ ID NO: 40.
In one embodiment, the sdAb comprises SEQ ID NO: 41.
In one embodiment, the sdAb comprises SEQ ID NO: 42.
In one embodiment, the sdAb comprises SEQ ID NO: 43.
In one embodiment, the sdAb comprises SEQ ID NO: 44.
In one embodiment, the sdAb comprises SEQ ID NO: 45.
In one embodiment, the sdAb comprises SEQ ID NO: 46.
In one embodiment, the sdAb comprises SEQ ID NO: 47.
In one embodiment, the sdAb comprises SEQ ID NO: 48.
In some embodiments, the BiKE is a sequence variant of the above BiKE having 80%, 90%, 95%, 98%, or 99% identity to one of the above-described BiKEs. In some embodiments, the variant retains substantially the same binding specificity as the parent molecule from which it is derived. In some embodiments the variant retains substantially the same binding affinity as the parent molecule from which it is derived.
BiKEs & TriKEs
In one embodiment, the multivalent antibody is a bispecific killer cell engager.
The term “BiKE” refers to a recombinant bispecific protein that has two linked variable regions from two different antibodies, one targeting a cell-surface molecule on natural killer (NK) cells (for example, CD16), and the other targeting antigens on the surface of disease cells, typically malignant cells. For example the BiKE may comprises two scFvs, two VHHs, or a combination thereof. The two are typically linked together by a short flexible linker. By binding to tumor antigens and NK cells simultaneously, BiKEs mediate NK-cell responses and killing of tumor cells.
In one embodiment, the cell-surface marker of the immune cell comprises a natural killer (NK) cell marker. In one embodiment, the NK cell marker comprises human CD16.
In one embodiment, the multivalent antibody is a trispecific killer cell engager (BiKE).
The term “TriKE” indicates at a BiKE that has been further modified to include another functionality. This term has been used to encompass various approaches. One approach involves inserting an intervening immunomodulatory molecule (a modified human IL-15 crosslinker) to promote NK cell activation, expansion, and/or survival (Vallera et al. IL-15 Trispecific Killer Engagers (TriKEs) Make Natural Killer Cells Specific to CD33+ Targets While Also Inducing In Vivo Expansion, and Enhanced Function. Clinical Cancer Research. 2012; 22(14): 3440-50). Other TriKE approaches are trispecific molecules that include three antibody variable regions: one targeting an NK cell receptor and two that target tumour-associated antigens (Gleason et al. Bispecific and Trispecific Killer Cell Engagers Directly Activate Human NK Cells Through CD16 Signaling and Induce Cytotoxicity and Cytokine Production. Mol Cancer Ther. 2012; 11(12): 2674-84). Yet other TriKE approaches target two NK cell receptors (e.g., CD16 and NKp46) and one tumour-associated antigen (Gauthier et al. Multifunctional Natural Killer Cell Engagers Targeting NKp46 Trigger Protective Tumor Immunity. Cell. 2019; 177(7): 1701-13).
In one embodiment, the multivalent antibody further comprises a cytokine for stimulating activation, expansion, and/or survival of NK cells. In one embodiment, the cytokine for stimulating expansion of NK cells is interleukin-15 (IL15), a variant thereof, or a functional fragment thereof.
In one embodiment, the multivalent antibody further comprises at least a third antigen-binding portion that binds to a second NK cell marker. In one embodiment, the second NK cell marker is human NKp46.
In one embodiment, the multivalent antibody further comprises at least a third antigen-binding portion that binds to a tumour-associated antigen. In some embodiment, the tumour-associated antigen is distinct from human EGFR.
In one embodiment, the third antigen-binding portion comprises a VHH, a VNAR, or an scVF.
In one embodiment, the second antigen-binding portion comprises a VHH.
In one embodiment, the third antigen-binding portion binds to human serum albumin. In such embodiment, the affinity for human serum albumin may contribute to stabilization/increased half-life.
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 amino acid sequence as set forth in SEQ ID NO: 2, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 3 (from NRC-sdAb021).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 4, a CDR2 amino acid sequence as set forth in SEQ ID NO: 5, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 6 (from NRC-sdAb022).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 7, a CDR2 amino acid sequence as set forth in SEQ ID NO: 8, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 9 (from NRC-sdAb023).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 10, a CDR2 amino acid sequence as set forth in SEQ ID NO: 11, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 12 (from NRC-sdAb024).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 13, a CDR2 amino acid sequence as set forth in SEQ ID NO: 14, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 15 (from NRC-sdAb025).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 16, a CDR2 amino acid sequence as set forth in SEQ ID NO: 17, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 18 (from NRC-sdAb026).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 amino acid sequence as set forth in SEQ ID NO: 20, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 21 (from NRC-sdAb027)
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 22, a CDR2 amino acid sequence as set forth in SEQ ID NO: 23, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 24 (from NRC-sdAb028).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 25, a CDR2 amino acid sequence as set forth in SEQ ID NO: 26, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 27 (from NRC-sdAb032).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 28, a CDR2 amino acid sequence as set forth in SEQ ID NO: 29, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 30 (NRC-sdAb033).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 31, a CDR2 amino acid sequence as set forth in SEQ ID NO: 32, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 33 (from NRC-VH54).
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 34.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 35.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 36.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 37.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 38.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 39.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 40.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 41.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 42.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 43.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 44.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 45.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 46.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 47.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 48.
In one embodiment, the sdAb comprises SEQ ID NO: 34.
In one embodiment, the sdAb comprises SEQ ID NO: 35.
In one embodiment, the sdAb comprises SEQ ID NO: 36.
In one embodiment, the sdAb comprises SEQ ID NO: 37.
In one embodiment, the sdAb comprises SEQ ID NO: 38.
In one embodiment, the sdAb comprises SEQ ID NO: 39.
In one embodiment, the sdAb comprises SEQ ID NO: 40.
In one embodiment, the sdAb comprises SEQ ID NO: 41.
In one embodiment, the sdAb comprises SEQ ID NO: 42.
In one embodiment, the sdAb comprises SEQ ID NO: 43.
In one embodiment, the sdAb comprises SEQ ID NO: 44.
In one embodiment, the sdAb comprises SEQ ID NO: 45.
In one embodiment, the sdAb comprises SEQ ID NO: 46.
In one embodiment, the sdAb comprises SEQ ID NO: 47.
In one embodiment, the sdAb comprises SEQ ID NO: 48.
In some embodiments, the BiKE or TriKE is a sequence variant of one of the above BiKEs and TriKEs having 80%, 90%, 95%, 98%, or 99% identity thereto. In some embodiments, the variant retains substantially the same binding specificity as the parent molecule from which it is derived. In some embodiments the variant retains substantially the same binding affinity as the parent molecule from which it is derived.
In one embodiment, there is provided a multivalent antibody comprising: a first antigen-binding portion, an amino acid linker comprising a polypeptide hinge from human CD8, and a second antigen-binding portion. In one embodiment, the polypeptide hinge from human CD8 comprises SEQ ID NO: 55. In one embodiment, the amino acid linker further comprises at least one G4S positioned N-terminal to the polypeptide hinge from human CD8, and at least one G4S positioned C-terminal to the polypeptide hinge from human CD8. In one embodiment, the amino acid linker is at least 47 amino acids in length, preferably is at least 52 amino acids in length, preferably at least 57 amino acids in length, more preferably at least 62 amino acids in length, even more preferably at least 67 amino acids in length. In one embodiment, the amino acid linker comprises, in N-terminal to C-terminal, direction SEQ ID NOs: 54, 55, and 62. In one embodiment, the first antigen-binding portion binds specifically to human EGFR. In one embodiment, the first antigen-binding portion is a VHH, VNAR, or an scVF. In one embodiment, the first antigen-binding portion is one the anti-EGFR sdAbs as described herein. In one embodiment, the second antigen-binding moiety binds specifically to a cell-surface marker of an immune cell. In one embodiment, he cell-surface marker of the immune cell comprises a T-cell marker. In one embodiment, the T-cell marker comprises human CD3. In one embodiment, the second antigen binding portion is a VHH, VNAR, or an scVF.
Table 7 lists example sequences for and modules of multivalent antibodies described herein, according to certain aspects and embodiments. For the example bispecific T-cell engager construct comprising antibody EGFR1 (sdAb021) (SEQ ID NO: 69):
For the purposes of experimentation and testing, the construct thereafter comprises positions 228-234, which correspond to a 6×His tag and two stop codons.
Recombinant Nucleic Acid Molecules
In aspect, there is provided a recombinant nucleic acid molecule encoding the multivalent antibody as defined herein. In one embodiment, nucleic acid is a vector. In one embodiment, the nucleic acid is DNA. In one embodiment, the nucleic acid is RNA. In one embodiment, the RNA is an mRNA. In one embodiment, the mRNA encodes for a therapeutic.
Compositions
In one aspect, there is provided a composition comprising a multivalent antibody as defined herein; together with an acceptable excipient, diluent or carrier. In one embodiment, the composition comprises a bispecific T-cell engager as herein defined. In one embodiment, the composition comprises a BiKE as herein defined. In one embodiment, the composition comprises a TriKE as herein defined. In one embodiment the composition is a pharmaceutical composition, and the excipient, diluent or carrier is a pharmaceutically acceptable excipient, diluent or carrier.
Uses & Methods
In one aspect, there is provided a use of the multivalent antibody as defined herein for treatment of a cancer.
In one aspect, there is provided a use of the multivalent antibody as defined herein for preparation of a medicament for treatment of a cancer.
In one aspect, there is provided the multivalent antibody as defined herein for use in treatment of a cancer.
In one aspect, there is provided a method of treating a cancer in subject comprising administering to the subject the multivalent antibody as defined herein.
In one embodiment, the cancer expresses EGFR. In one embodiment, the cancer aberrantly expresses EGFR normally absent in corresponding healthy tissue of origin and/or surrounding tissue. In one embodiment, the cancer expresses high levels of EGFR compared to corresponding healthy tissue of origin and/or surrounding tissue. In one embodiment, the cancer is selected from the group consisting of (but not limited to) lung cancer, pancreatic cancer, gastric cancer, brain cancer (such as glioblastoma), neuroendocrine cancer, head and neck cancer, bladder cancer, breast cancer, colorectal cancer, cervical cancer, and endometrial cancer.
Chimeric Antibody Receptors & Related Embodiments
In one aspect, there is provided a chimeric antibody receptor (CAR), which binds to human EGFR, comprising the VHH sdAb as defined herein.
“Chimeric antigen receptors” are receptor proteins engineered to give T cells the new ability to target a specific protein. The receptors are chimeric because they combine both antigen-binding and T-cell activating functions into a single receptor (see Stoiber et al. Limitations in the Design of Chimeric Antigen Receptors for Cancer Therapy. Cells. 2012; 8(5): 472 and van der Stegen et al. The pharmacology of second-generation chimeric antigen receptors. Nat Rev Drug Discov. 2019; 14(7): 499-509).
In one embodiment, the CAR comprises, in N-terminal to C-terminal direction:
The term “polypeptide hinge” used herein generally means any oligo- or polypeptide that functions to link the extracellular ligand-binding domain to the transmembrane domain. In particular, hinge region are used to provide more flexibility and accessibility for the extracellular ligand-binding domain. A hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. Hinge region may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or may be an entirely synthetic hinge sequence.
In one embodiment, the polypeptide hinge comprises a CD8 hinge domain. In one embodiment, the CD8 hinge domain comprises SEQ ID NO: 55 or C-terminal fragment thereof comprising at least 22 C-terminal amino acids of SEQ ID NO: 55. In one embodiment, the CD8 hinge domain comprises SEQ ID NO: 55. In one embodiment, the CD8 hinge domain is at least 80% identical to SEQ ID NO: 55. In one embodiment, hinge domain is at least 90% identical to SEQ ID NO: 55. In one embodiment, the hinge domain is at least 95% identical to SEQ ID NO: 55. In one embodiment, the hinge domain is at least 98% identical to SEQ ID NO: 55.
The hinge may comprise a fragment of SEQ ID NO: 55. The hinge may comprise an C-terminal fragment of SEQ ID NO: 55. The hinge may comprise the C-terminal 22 amino acids of SEQ ID NO: 55 (SEQ ID NO: 57). The hinge may comprise the C-terminal 23 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 24 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 25 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 26 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 27 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 28 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 29 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 30 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 31 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 32 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 33 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 34 amino acids of SEQ ID NO: 55 (SEQ ID NO: 56). The hinge may comprise the C-terminal 35 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 36 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 37 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 38 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 39 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 40 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 41 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 42 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 43 amino acids of SEQ ID NO: 55. The hinge may comprise the C-terminal 44 amino acids of SEQ ID NO: 55. In one embodiment, the hinge comprises a sequence that is at least 80% identical to one of the aforementioned C-terminal fragments. In one embodiment, the hinge comprises a sequence that is at least 90% identical to one of the aforementioned C-terminal fragments. In one embodiment, the hinge comprises a sequence that is at least 95% identical to one of the aforementioned C-terminal fragments. In one embodiment, the hinge comprises a sequence that is at least 98% identical to one of the aforementioned C-terminal fragments.
In one embodiment, the polypeptide hinge comprises SEQ ID NO: 71 or a C-terminal fragment thereof comprising at least 22 amino acids.
In some embodiments, hinge length may be adjusted to achieve a desired property, as described herein. While, C-terminal fragments are discussed herein, it is also envisaged that N-terminal fragments or internal truncations (deletions) of SEQ ID NO: 71 could also be made and tested to determine which possess the desired property. These fragments or internal truncations may comprise at least 22 amino acids of SEQ ID NO: 71. They may comprise at least 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 amino acids of SEQ ID NO: 71.
In one embodiment, the hinge length may be selected to achieve maximum signaling, i.e. maximum activity toward target cells. Signaling can be assayed in vitro using, e.g., EGFR-high SKOV3 cells and, e.g., according to assays described herein. For example, a hinge comprising from 40 and 52 C-terminal amino acids of human of SEQ ID NO: 71 may achieve maximum signalling, e.g., for a CAR comprising a sdAb comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO:3; or wherein the CAR comprises SEQ ID NO: 34 (sdAb021). In some embodiments, a CAR construct comprising a hinge conveying these properties would be suited to targeting strategies that incorporate additional mechanisms to downregulate on-target off tumour responses. For example, these strategies may comprise Natural Killer-based cellular immunotherapy or Boolean-gated CAR receptors. The hinge may comprise 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 C-terminal amino acids of SEQ ID NO: 71.
In one embodiment, the hinge length may be selected to maximize selectivity for target cells, i.e. discrimination for EGFR-high cells over EGFR-low cells. This may be tested, e.g., according to the assays described herein using, e.g., EGFR-high SKOV3 cells vs. EGFR-low MCF7 cells. For example, a hinge comprising from 34 to 39 C-terminal amino acids of human of SEQ ID NO: 71 may achieve maximum signalling, e.g., for a CAR comprising a sdAb comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO:3; or wherein the CAR comprises SEQ ID NO: 34 (sdAb021). In some embodiments, a CAR construct comprising a hinge conveying this property would be suited to “single CAR” applications, for example applications in which no other CAR or cellular regulation strategy is used. The hinge may comprise 34, 35, 36, 37, 38, or 39 C-terminal amino acids of SEQ ID NO: 71.
In one embodiment, the hinge length may be selected to achieve low activity towards target cells, i.e. some binding to EGFR-high target cells with negligible to no detectable signaling. This may be tested, e.g., according the assays described herein using, e.g., EGFR-high SKOV3 cells. For example, a hinge comprising from 22 to 33 C-terminal amino acids of human of SEQ ID NO: 55 may achieve maximum signalling, e.g., for a CAR comprising a sdAb comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO:3; or wherein the CAR comprises SEQ ID NO: 34 (sdAb021). In some embodiments, a CAR construct comprising a hinge conveying this properties would be suited for multi-antigen targeting CAR applications that incorporate additional targeting elements. These may include, but are not limited to, tandem CAR constructs containing multiple antigen binding domains, bi-cistronic CAR constructs wherein multiple CAR receptors are encoded in a single viral construct, co-transduction of CAR viruses targeting different antigens, or co-administration of separate CAR-T product which target different antigens. The hinge may comprises 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 C-terminal amino acids of SEQ ID NO: 71.
The term “transmembrane domain” indicates a polypeptide having the ability to span a cell membrane and thereby link the extracellular portion of the CAR (which comprises the EGFR-binding portion) to the intracellular portion responsible for signaling. Commonly used transmembrane domains for CARs have been derived from CD4, CD8α, CD28 and CD3ζ.
In one embodiment, the transmembrane domain is a CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises SEQ ID NO: 58. In one embodiment, the transmembrane domain is at least 80% identical to SEQ ID NO: 58. In one embodiment, the transmembrane domain is at least 90% identical to SEQ ID NO: 58. In one embodiment, the transmembrane domain is at least 95% identical to SEQ ID NO: 58. In one embodiment, the transmembrane domain is at least 98% identical to SEQ ID NO: 58.
The term “cytoplasmic domain” (also termed a “signal transduction domain”) refers to the intracellular portion of the CAR that is responsible for intracellular signaling following the binding of extracellular ligand binding domain to the target resulting in the activation of the immune cell and immune response. In other words, cytoplasmic domain is responsible for the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be a cytolytic activity or helper activity including the secretion of cytokines. Thus, the term “cytoplasmic domain” refers to the portion of a protein which transduces the effector signal and directs the cell to perform a specialized function. It is common for such cytoplasmic domains to comprise a co-stimulatory domain in addition to a signaling domain.
The term “signaling domain” refers to the portion of a protein which transduces the effector signal and directs the cell to perform a specialized function. Examples of signal transducing domain for use in a CAR can be the cytoplasmic sequences of the T cell receptor and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivate or variant of these sequences and any synthetic sequence that has the same functional capability. Signal transducing domain comprises two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal. Primary cytoplasmic signaling sequence can comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. ITAMs are well defined signaling motifs found in the intracytoplasmic tail of a variety of receptors that serve as binding sites for syk/zap70 class tyrosine kinases. Non-limiting examples of signaling domains used in the invention can include those derived from TCRzeta, common FcR gamma (FCERIG), Fcgamma Rlla, FcRbeta (Fc Epsilon Rib), FcRepsilon, CD3 zeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, CD66d, DAP10, or DAP12. In a preferred embodiment, the signaling transducing domain of the CAR can comprise the CD3zeta signaling domain.
In one embodiment, the signaling domain is a CD3-zeta signaling domain. In one embodiment, the CD3-zeta signaling domain comprises SEQ ID NO: 60. In one embodiment, the signaling domain is at least 80% identical to SEQ ID NO: 60. In one embodiment, the signaling domain is at least 90% identical to SEQ ID NO: 60. In one embodiment, the signaling domain is at least 95% identical to SEQ ID NO: 60. In one embodiment, the signaling domain is at least 98% identical to SEQ ID NO: 60.
The term “co-stimulatory domain” refers to the cognate binding partner on a T-cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the cell, such as, but not limited to proliferation. Co-stimulatory molecules include, but are not limited to, an MHC class I molecule, BTLA and Toll ligand receptor. Examples of costimulatory molecules include CD27, CD28, 4-1 BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI Id, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDlIc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG2D or a combination thereof.
In one embodiment, the co-stimulatory domain is a 4-1BB co-stimulatory domain. In one embodiment, the 4-1BB signal transduction domain comprises SEQ ID NO: 59. In one embodiment, the co-stimulatory domain is at least 80% identical to SEQ ID NO: 59. In one embodiment, the co-stimulatory domain is at least 90% identical to SEQ ID NO: 59. In one embodiment, the co-stimulatory domain is at least 95% identical to SEQ ID NO: 59. In one embodiment, the co-stimulatory domain is at least 98% identical to SEQ ID NO: 59.
In one embodiment, CAR further comprises a flexible amino acid linker between the sdAb and the polypeptide hinge. In one embodiment, the amino acid linker comprises SEQ ID NO: 62. In one embodiment, the amino acid linker is at least 80% identical to SEQ ID NO: 62. In one embodiment, the amino acid linker is at least 90% identical to SEQ ID NO: 62. In one embodiment, the amino acid linker is at least 95% identical to SEQ ID NO: 62. In one embodiment, the amino acid linker is at least 98% identical to SEQ ID NO: 62.
In one embodiment, the CAR further comprises a signal peptide.
In one embodiment, the signal peptide is a signal peptide from human CD28. In one embodiment, the signal peptide from human CD28 comprises SEQ ID NO: 53. In one embodiment, the signal peptide is at least 80% identical to SEQ ID NO: 53. In one embodiment, the signal peptide is at least 90% identical to SEQ ID NO: 53. In one embodiment, the signal peptide is at least 95% identical to SEQ ID NO: 53. In one embodiment, the signal peptide is at least 98% identical to SEQ ID NO: 53.
In one embodiment, the CAR is encoded by SEQ ID NO: 62.
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 1, a CDR2 amino acid sequence as set forth in SEQ ID NO: 2, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 3 (NRC-sdAb021).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 4, a CDR2 amino acid sequence as set forth in SEQ ID NO: 5, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 6 (NRC-sdAb022).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 7, a CDR2 amino acid sequence as set forth in SEQ ID NO: 8, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 9 (NRC-sdAb023).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 10, a CDR2 amino acid sequence as set forth in SEQ ID NO: 11, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 12 (NRC-sdAb024).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 13, a CDR2 amino acid sequence as set forth in SEQ ID NO: 14, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 15 (NRC-sdAb025).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 16, a CDR2 amino acid sequence as set forth in SEQ ID NO: 17, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 18 (NRC-sdAb026).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 19, a CDR2 amino acid sequence as set forth in SEQ ID NO: 20, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 21 (NRC-sdAb027)
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 22, a CDR2 amino acid sequence as set forth in SEQ ID NO: 23, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 24 (NRC-sdAb028).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 25, a CDR2 amino acid sequence as set forth in SEQ ID NO: 26, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 27 (NRC-sdAb032).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 28, a CDR2 amino acid sequence as set forth in SEQ ID NO: 29, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 30 (NRC-sdAb033).
In one embodiment, the sdAb comprises a CDR1 amino acid sequence as set forth in SEQ ID NO: 31, a CDR2 amino acid sequence as set forth in SEQ ID NO: 32, and a CDR3 amino acid sequence as set forth in SEQ ID NO: 33 (NRC-VH54).
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 34.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 35.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 36.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 37.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 38.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 39.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 40.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 41.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 42.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 43.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 44.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 45.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 46.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 47.
In one embodiment, the sdAb comprises a CDR1, CDR2, and CDR3 of the sdAb sequence set forth in SEQ ID NO: 48.
In one embodiment, the sdAb comprises SEQ ID NO: 34.
In one embodiment, the sdAb comprises SEQ ID NO: 35.
In one embodiment, the sdAb comprises SEQ ID NO: 36.
In one embodiment, the sdAb comprises SEQ ID NO: 37.
In one embodiment, the sdAb comprises SEQ ID NO: 38.
In one embodiment, the sdAb comprises SEQ ID NO: 39.
In one embodiment, the sdAb comprises SEQ ID NO: 40.
In one embodiment, the sdAb comprises SEQ ID NO: 41.
In one embodiment, the sdAb comprises SEQ ID NO: 42.
In one embodiment, the sdAb comprises SEQ ID NO: 43.
In one embodiment, the sdAb comprises SEQ ID NO: 44.
In one embodiment, the sdAb comprises SEQ ID NO: 45.
In one embodiment, the sdAb comprises SEQ ID NO: 46.
In one embodiment, the sdAb comprises SEQ ID NO: 47.
In one embodiment, the sdAb comprises SEQ ID NO: 48.
In some embodiments, the CAR is a sequence variant of one of the above CARs having 80%, 90%, 95%, 98%, or 99% identity thereto. In some embodiments, the variant retains substantially the same binding specificity as the parent molecule from which it is derived. In some embodiments the variant retains substantially the same binding affinity as the parent molecule from which it is derived.
Table 7 lists example sequences for and modules of multivalent antibodies and CARs described herein, according to certain aspects and embodiments.
For the example CAR construct comprising antibody EGFR1 (sdAb021) (SEQ ID NO: 66):
Nucleic Acids & Vectors
In one aspect, there is provided a recombinant nucleic acid molecule encoding the CAR as defined herein.
In one aspect, there is provided a vector comprising the recombinant nucleic acid molecule as defined herein. In one embodiment, the vector is a viral vector. In one embodiment, the viral vector is a lentivirus vector.
Viral Particles
In one aspect, there is provided a recombinant viral particle comprising the recombinant nucleic acid as defined herein. In one embodiment, the recombinant viral particle is a recombinant lentiviral particle.
Cells
In one aspect, there is provided a cell comprising the recombinant nucleic acid molecule as defined herein. In one embodiment, the recombinant nucleic acid molecule is DNA. In one embodiment, the recombinant nucleic acid molecule is RNA. In one embodiment, the RNA is mRNA. In one embodiment, the mRNA encodes a therapeutic.
In one embodiment, there is provided an engineered cell expressing at the cell surface membrane the CAR as defined herein. In one embodiment, the engineered cell is an immune cell. In one embodiment, the immune cell is a T-lymphocyte or is derived from T-lymphocytes.
Use & Methods
“CAR-T” cell therapy uses T cells engineered with CARs for cancer therapy. The premise of CAR-T immunotherapy is to modify T cells to recognize disease cells, typically cancer cells, in order to more effectively target and destroy them. Generally, T are genetically altered to express a CAR, and these cells are infused into a patient to attack their tumors. CAR-T cells can be either derived from T cells in a patient's own blood (autologous) or derived from the T cells of another healthy donor (allogeneic).
In one aspect, there is providing a use of the nucleic acid, vector, or viral particle as described herein for preparation of cells for CAR-T.
In one aspect, there is providing a method of preparing cells for CAR-T comprising contacting a T-cell with the viral particle as described herein. In one embodiment, the T-cell is from a donor. In one embodiment, the T-cell is from a patient.
In one aspect, there is providing a method of preparing cells for CAR-T comprising introducing into a T-cell the nucleic acid or vector as described herein. In one embodiment, the T-cell is from a donor. In one embodiment, the T-cell is from a patient.
In one aspect, there is provided a use of the CAR or of the engineered cell as described herein for treatment of a cancer.
In one embodiment, the method further comprises an initial step of obtaining cells from a patient or donor and introducing the recombinant nucleic acid molecule or vector encoding the CAR, as described herein.
In one embodiment, the method further comprises an initial step of obtaining cells from a patient or donor and contacting the cells with the viral particle, as described herein.
In one aspect, there is provided a use of the CAR or of the engineered cell as described herein for preparation of a medicament treatment of a cancer.
In one aspect, there is provided the CAR or the engineered cell as described herein for use in treatment of a cancer.
In one aspect there is provided a method of treating a cancer in a subject, comprising administering to the subject the engineered cell as defined herein.
In one embodiment, the cancer expresses EGFR. In one embodiment, the cancer aberrantly expresses EGFR normally absent in corresponding healthy tissue of origin and/or surrounding tissue. In one embodiment, the cancer expresses high levels of EGFR compared to corresponding healthy tissue of origin and/or surrounding tissue. In one embodiment, the cancer is selected from the group consisting of (but not limited to) lung cancer, pancreatic cancer, gastric cancer, brain cancer (such as glioblastoma), neuroendocrine cancer, head and neck cancer, bladder cancer, breast cancer, colorectal cancer, cervical cancer, and endometrial cancer.
The following Examples outline embodiments of the invention and/or studies conducted pertaining to the invention. While the Examples are illustrative, the invention is in no way limited the following exemplified embodiments.
Abbreviations: Abs, antibodies; AF488, Alexa Fluor® 488; APC, allophycocyanin; BSA, bovine serum albumin; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GPCR, G protein-coupled receptor; HBSS, Hank's Balanced Salt Solution; HRP, horseradish peroxidase; IMAC, immobilized metal affinity chromatography; LCIB, live cell imaging buffer; PBS, phosphate-buffered saline; PE, R-phycoerythrin; RU, resonance unit; sdAb, single-domain antibody; SPR, surface plasmon resonance; TMB, 3,3′,5,5′-tetramethylbenzidine; VHH, variable domain of camelid heavy chain-only antibody.
Generation and Characterization of Single-Domain Antibodies
In this Example, Camelid single-domain antibodies are raised to EGFR by DNA immunization.
Background
DNA immunization of large outbred animals is generally recognized as inconsistent and poorly effective in eliciting humoral immune responses. The mechanisms underlying this difficulty are thought to involve low rates of plasmid uptake and antigen expression, potentially relating to concentration effects in peripheral tissue. Studies have examined DNA immunization of camelid species, with generally poor results. Here, the hypothesis was tested that high-affinity and functional camelid sdAbs could be produced against a model antigen, EGFR, using DNA immunization alone. Herein is described the comprehensive in vitro characterization of a panel of sdAbs generated in this manner, which were dramatically superior to those previously isolated using protein immunization.
Materials and Methods
Antibodies and Reagents
Recombinant 6×His-tagged human EGFRvIII was produced by transient transfection of HEK293-6E cells as previously described [17] and purified by immobilized metal affinity chromatography (IMAC) followed by a final size exclusion chromatography polishing step to remove aggregates. Recombinant 6×His-tagged human EGFR ectodomain was from Genscript (Cat. No. Z03194; Piscataway, NJ), recombinant in vivo biotinylated 6×His-tagged human EGFRvIII ectodomain was from ACROBiosystems (Cat. No. EGR-H82E0; Newark, DE) and recombinant streptavidin was from Thermo Fisher Scientific (Waltham, MA). Human EGFR-Fc fusion protein was from Genscript (Cat. No. Z03381), and rhesus and mouse EGFR-Fc fusion proteins were from Sino Biological (Cat. Nos. 90317-K02H and 51091-M02H; Beijing, China). Horseradish peroxidase (HRP)-conjugated goat polyclonal anti-llama IgG was from Cedarlane Laboratories (Cat. No. A160-100P; Burlington, Canada), HRP-conjugated goat polyclonal anti-human IgG was from Sigma-Aldrich (St. Louis, MO) and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was from Mandel Scientific (Guelph, Canada). Bovine serum albumin (BSA) and Tween-20 were from Sigma-Aldrich, and all cell culture reagents were from Thermo Fisher. Mouse monoclonal anti-c-Myc IgG was from Santa Cruz Biotechnology (clone 9E10, Cat. No. sc-40; Dallas, TX), allophycocyanin (APC)-conjugated goat polyclonal anti-mouse IgG was from Thermo Fisher (Cat. No. A865), Alexa Fluor® 488 (AF488)-conjugated donkey anti-human IgG was from Jackson ImmunoResearch (Cat. No. 709546098; West Grove, PA) and R-phycoerythrin (PE)-conjugated streptavidin was from Thermo Fisher (Cat. No. S866). Erlotinib was from Sigma-Aldrich, recombinant human epidermal growth factor (EGF) and mouse monoclonal Ab against β-actin were from Genscript (Cat. Nos. Z00333 and A00702), rabbit polyclonal Ab against phospho-EGFR (Tyr1068) was from Cell Signaling Technology (Cat. No. 2234; Danvers, MA), mouse monoclonal Ab against human EGFR was a generous gift from Anne Marcil (National Research Council Canada, Montreal, Canada) and cetuximab was a generous gift from Yves Durocher (National Research Council Canada, Montreal, Canada). HRP-conjugated donkey polyclonal anti-mouse IgG was from Jackson ImmunoResearch (Cat. No. 715036150) and HRP-conjugated goat polyclonal anti-rabbit IgG was from Cedarlane Laboratory (Cat. No. CLCC43007). SuperSignal™ West Pico PLUS chemiluminescent substrate was from Thermo Fisher.
Llama Immunization
A male llama (Lama glama) was immunized by biolistic transfection using a Helios® gene gun system (Bio-Rad, Hercules, CA) followed by intradermal injection using a DERMOJET device (AKRA DERMOJET, Pau, France). Two pTT5 vectors [17] encoding either soluble human EGFRvII (UniProt P00533: residues 1-29/Gly/297-645) or membrane-tethered human EGFRvII (UniProt P00533: residues 1-29/Gly/297-668) were purified from overnight cultures of Escherichia coli DH5α cells using a QIAGEN® Plasmid Maxi Kit (Qiagen, Hilden, Germany). Briefly, 50 mg of gold particles were coated with 100 μL of 0.05 M spermidine, then vortexed and sonicated. An equimolar mixture of both pTT5 vectors (50 pg each; 100 pg total DNA in 100 μL ultrapure water) was added to the spermidine-coated gold particles, then 100 μL of 1 M CaCl2 was added dropwise to the mixture. After incubating for 10 min at room temperature, the gold particles were pelleted in a microfuge, washed three times with 100% ethanol and resuspended in 6 ml of 100% ethanol containing 0.05 mg/ml polyvinylpyrrolidone. The DNA-gold solution was dried onto the inner walls of two 30-inch lengths of gold-coat tubing under nitrogen flow, then the tubing was cut into 0.5-inch lengths.
The llama was immunized six times (weeks 0, 2, 4, 6, 9 and 12) by biolistic transfection; each immunization consisted of 12 bombardments administered at 600 PSI to shaved sites on the neck and hind limb (10 pg of total DNA per immunization). Thereafter, four additional immunizations (weeks 16, 20, 24 and 28) were administered by intradermal injection of 1 mg (1 mg/ml) of DNA using a DERMOJET device. Serum titration ELISA was conducted as described previously [18], and binding was detected using HRP-conjugated polyclonal goat anti-llama IgG. Experiments involving animals were conducted using protocols approved by the National Research Council Canada Animal Care Committee and in accordance with the guidelines set out in the OMAFRA Animals for Research Act, R. S. O. 1990, c. A.22.
Construction and Panning of Phage-Displayed VHH Library
A phage-displayed VHH library was constructed from the peripheral blood lymphocytes of the immunized llama as described previously [18]. Briefly, peripheral blood mononuclear cells were purified by density gradient centrifugation from blood obtained 5 days following the third and the final DERMOJET immunizations. Total RNA was extracted from −5×107 cells from each time point using a PureLink™ RNA Mini Kit (Thermo Fisher) and cDNA was reverse transcribed using qScript® cDNA supermix containing random hexamer and oligo(dT) primers (Quanta Biosciences, Gaithersburg, MD). VHH genes were amplified using semi-nested PCR and cloned into the phagemid vector pMED1; the final library had a size of 3×107 independent transformants and an insert rate of −75%. Phage particles were rescued from the library using M13K07 helper phage and panned against microplate-adsorbed human EGFRvIII for three rounds with triethylamine elution as described previously [18]. A second independent library selection was carried out in the same manner, except that the target was streptavidin-captured biotinylated EGFRvIII.
Expression of VHHs and VHH-Fc Fusions
VHH DNA sequences were cloned into the pSJF2H expression vector and monomeric VHHs tagged C-terminally with c-Myc and 6×His were purified from the periplasm of E. coli TG1 cells by IMAC as previously described [18]. In addition, in vivo biotinylated monomeric VHHs were produced by co-transformation of E. coli BL21 (DE3) cells with two vectors encoding (i) VHHs C-terminally tagged with a biotin acceptor peptide and 6×His and (ii) the biotin ligase BirA and purified by IMAC [19]. Bivalent VHH-human IgG1 Fc fusions were produced by transient transfection of HEK293-6E cells followed by protein A affinity chromatography as previously described [17]. Heterodimeric biparatopic VHH-Fc fusions were produced by co-transfection of HEK293-6E cells with two pTT5 vectors encoding (i) NRC-sdAb032-Fc tagged C-terminally with 6×His and (ii) a second untagged VHH-Fc. The heterodimeric Ab was purified by sequential protein A affinity chromatography and IMAC and eluted using a linear 0→0.5 M imidazole gradient over 20 column volumes to separate species bearing one or two 6×His tags. VHHs and VHH-Fcs were dialyzed against or buffer-exchanged into phosphate-buffered saline (PBS), pH 7.4.
ELISA and EGF-Competition ELISA
Wells of NUNCO MaxiSorp™ microtiter plates (Thermo Fisher) were coated overnight at 4° C. with 2 pg/ml streptavidin in 100 μL of PBS, pH 7.4. The wells were blocked with 200 μL of PBS containing 1% (w/v) BSA for 1 h at 37° C. and then biotinylated VHHs [10 pg/ml in 100 μL of PBS containing 1% BSA and 0.1% (v/v) Tween-20] were captured for 30 min at room temperature. The wells were washed 5× with PBS containing 0.1% Tween-20 and then incubated with human EGFR-Fc (500 ng/ml in 100 μL of PBS containing 1% BSA and 0.05% Tween-20) in the presence or absence of EGF (17 pg/ml) for 1 h at room temperature. The wells were washed 5× again and incubated with HRP-conjugated goat anti-human IgG (1 pg/ml in 100 μL of PBS containing 1% BSA and 0.05% Tween-20) for 1 h at room temperature. After a final wash (5× with PBS containing 0.1% Tween-20), the wells were developed with TMB substrate, stopped with 1 M H2SO4 and the absorbance at 450 nm was measured using a Multiskan™ FC photometer (Thermo Fisher).
Surface Plasmon Resonance
Prior to surface plasmon resonance (SPR) analyses, monomeric VHHs were purified by preparative size exclusion chromatography using a Superdex™ 75 10/300 GL column (GE Healthcare, Piscataway, NJ) connected to an AKTA FPLC protein purification system (GE Healthcare). In the first SPR experiment, multi-cycle kinetic analyses were performed on a Biacore™ 3000 instrument (GE Healthcare) at 25° C. in HBS-EP buffer [10 mM HEPES, pH 7.4, containing 150 mM NaCl, 3 mM EDTA and 0.005% (w/v) surfactant P20]. Approximately 1304-2158 and 741 resonance units (RUs), respectively, of recombinant human EGFR and EGFRvIII ectodomains were immobilized on a CM5 sensor chip (GE Healthcare) in 10 mM acetate buffer, pH 4.5, using an amine coupling kit (GE Healthcare). An ethanolamine-blocked flow cell served as the reference. Monomeric VHHs at concentrations ranging from 0.1 to 100 nM were injected over the EGFR and EGFRvIII surfaces in HBS-EP buffer at a flow rate of 20 μL/min. For NRC-sdAb032 only, the VHH (212 RUs) was immobilized on a CM5 sensor chip by amine coupling and 0.5-50 nM recombinant human EGFR ectodomain was injected over the VHH surface in HBS-EP buffer at a flow rate of 20 μL/min. Contact times were 180-300 s and dissociation times were 300-600 s. The EGFR, EGFRvIII and NRC-sdAb032 surfaces were regenerated using a 10 s pulse of 10 mM glycine, pH 1.5.
In the second SPR experiment, single-cycle kinetic analyses were performed on a Biacore™ T200 instrument (GE Healthcare) at 25° C. in HBS-EP buffer. Approximately 618, 1051 and 600 RUs of human, rhesus and mouse EGFR-Fc, respectively, were immobilized on three flow cells of a Series S Sensor Chip CM5 (GE Healthcare) in 10 mM acetate buffer, pH 4.5, using an amine coupling kit. An ethanolamine-blocked flow cell served as the reference. Monomeric VHHs at concentrations ranging from 0.6 to 50 nM were injected over the EGFR surfaces in HBS-EP buffer at a flow rate of 40 μL/min. The contact time was 180 s and the dissociation time was 600 s. The EGFR surfaces were regenerated using a 10 s pulse of 10 mM glycine, pH 1.5.
Epitope-binning experiments were performed essentially as described above on a Biacore™ 3000 instrument, except that 3383 RUs of human EGFR-Fc were immobilized on a CM5 sensor chip. A single VHH (or cetuximab) at a concentration equivalent to 25×KD was injected at 40 μL/min with a contact time of 150 s to saturate the EGFR surface. The second injection consisted of the same VHH (or cetuximab) in the presence of 25× the KD concentration of a second VHH. All data were analyzed by fitting to a 1: 1 interaction model using BIAevaluation 4.1 software (GE Healthcare).
Flow Cytometry and Mirrorball® Assays
MDA-MB-468 and MCF7 cells were cultured at 37° C. in a humidified 5% C02 atmosphere in T75 flasks containing RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, 100 g/ml streptomycin and 250 ng/ml amphotericin B. For flow cytometry experiments, cells were grown to 70-80% confluency, dissociated using trypsin-EDTA solution, washed in PBS and then resuspended in PBS containing 1% BSA. Approximately 1×105 cells were stained sequentially on ice for 30 min with (i) 20 pg/ml of each VHH, (ii) 5 pg/ml of mouse anti-c-Myc IgG and (iii) 5 pg/ml of APC-conjugated goat anti-mouse IgG. The cells were washed with PBS in between each staining step, and after a final wash, data were acquired on a BD FACSCanto™ instrument (BD Biosciences, San Jose, CA).
For Mirrorball® experiments, cells were dissociated from flasks using Accutase® solution, washed in Hank's Balanced Salt Solution (HBSS) and then ˜5000 cells in growth medium were plated in each rat tail collagen-coated well of a Nunc™ MicroWell 96-well optical bottom plate (Thermo Fisher). After incubating at 37° C./5% C02 for 24 h, the cells were washed with HBSS and Abs [biotinylated VHHs, VHH-Fcs or cetuximab, serially diluted in live cell imaging buffer (LCIB) containing 1% BSA] were added to wells for 2 h at 4° C. The cells were washed with LCIB and secondary detection reagents (40 pg/ml PE-conjugated streptavidin or 30 pg/ml AF488-conjugated donkey anti-human IgG in LCIB containing 1% BSA) were added as appropriate to each well for 1 h at 4° C. The cells were washed with LCIB and stained with 1 M DRAQ5™ for 10 min at 4° C. After a final wash with LCIB, data were acquired on a Mirrorball® microplate cytometer (TTP Labtech, Melbourn, U.K.) and analyzed using Cellista software (TTP Labtech).
EGFR Phosphorylation Assay
Approximately 2×105 MDA-MB-468 cells were seeded in wells of 12-well tissue culture plates and then starved in serum-free RPMI-1640 medium overnight. The next day, the medium was replaced with RPMI-1640 containing 1% BSA and various concentrations of erlotinib or Abs (VHH-Fcs or cetuximab). After 30 min at 37° C., EGF was added to a final concentration of 25 nM and incubated for a further 15 min. The cells were cooled immediately on ice, washed twice with PBS and then scraped in 100 ml Laemmli buffer. Cell lysates (5 ml) were electrophoresed on 4-20% Mini-Protean® TGX™ precast gels (Bio-Rad Laboratories) and transferred to polyvinylidene fluoride membranes using the semi-dry method. Western blotting was performed as previously described [6]. Briefly, membranes were blocked overnight at 4° C. with 2% BSA in PBS, then sequentially incubated for 1 h at room temperature with (i) primary Abs (anti-phospho-EGFR, anti-EGFR or anti-β-actin, all diluted 1: 1500 in PBS containing 1% BSA and 0.1% Tween-20) and (ii) secondary Abs (HRP-conjugated donkey anti-mouse IgG or goat anti-rabbit IgG, diluted 1: 3000 in PBS containing 1% BSA and 0.1% Tween-20). The membranes were washed extensively with PBS containing 0.1% Tween-20 following incubations with primary and secondary Abs. The blots were developed using enhanced chemiluminescence and imaged using a Molecular Imager® Gel Doc™ XR+ System (Bio-Rad Laboratories). Band densitometry analysis was conducted using ImageJ version 1.52.
VHH Humanization
VHHs were humanized by alignment with human IGHV3-30*01 and IGHJ1-1*01 amino acid sequences. For each VHH, three humanized variants were designed representing a spectrum of increasing homology to the human germline: (i) variant H1, in which all FR sequences were reverted to the human consensus excepting residues located within five positions of an FR-CDR boundary; (ii) variant H2, in which all FR sequences were reverted to the human consensus excepting residues located within two positions of an FR-CDR boundary and (iii) variant H3, in which all FR sequences were fully human. CDR residues as well as FR2 positions 42 and 52 (IMGT numbering) were left unaltered in all variants.
Results and Discussion
Llama DNA Immunization Using Gene Gun and DERMOJET
A llama was immunized with DNA encoding human EGFRvIII six times every 2-3 weeks by biolistic transfection using a gene gun. No polyclonal serum Ab response was evident in the animal following the six gene gun immunizations, but boosting three times by intradermal injection using a DERMOJET device elicited serum Abs against recombinant EGFR with a half-maximal titer of ˜1: 5000 (
VHHs Elicited by DNA Immunization Targeted Five Unique EGFR Epitopes Including an Epitope Overlapping that of Cetuximab
A phage-displayed VHH library was constructed from the peripheral blood lymphocytes of the immunized llama and VHHs were isolated by panning against either plate-adsorbed EGFRvIII or streptavidin-captured biotinylated EGFRvIII. Ten unique VHHs falling into three sequence families were identified in the panning against plate-adsorbed EGFRvIII (NRC-sdAb021-NRC-sdAb030; Table 1).
aIMGT framework definitions
All of these VHHs as well as two others (NRC-sdAb032 and NRC-sdAb033) were identified by panning against streptavidin-captured biotinylated EGFRvIII. The VHHs had monovalent binding affinities for recombinant human EGFR, ranging from 1 to 40 nM as measured by SPR, and, despite immunization with DNA encoding EGFRvIII, all showed identical binding to EGFR and EGFRvIII (
1Determined by amine-coupling NRC-sdAb032 and flowing recombinant EGFR ectodomain.
2No difference was observed in ELISA binding to human EGFR or EGFRvIII (data not shown); n.d., not determined.
In contrast with a previous report, the EGFR-binding affinity of EG2 was measured (a VHH raised by immunization with the recombinant EGFRvIII ectodomain) as ˜15-20 nM, not 55 nM [4]. The VHHs showed a variety of cross-species reactivity patterns (Tables 3A and 3B, and
EG2 VHH bound only human EGFR, NRC-sdAb022-family VHHs and NRC-sdAb032 bound human and rhesus EGFR with similar affinities, and NRC-sdAb029-family VHHs and NRC-sdAb033 bound human, rhesus and mouse EGFR with similar affinities (
Despite their broadly similar monovalent affinities for recombinant EGFR (1-40 nM), the VHHs showed significant variability in their ability to recognize EGFR-positive tumor cell lines. Flow cytometry showed that four of five epitope bins targeted by the VHH monomers were accessible on native cell-surface EGFR; neither binding of NRC-sdAb029 nor, surprisingly, binding of EG2 to MDA-MB-468 cells was detectable at the single concentration (20 mg/ml, equivalent to ˜1.3 mM) used in this assay (
NRC-sdAb032, a VHH Elicited by DNA Immunization, Inhibited EGFR Signaling with Potency Similar to Cetuximab
The ability of all of the VHH-Fcs showing significant binding to EGFR-positive tumor cells (NRC-sdAb022-Fc, NRC-sdAb028-Fc, NRC-sdAb032-Fc and NRC-sdAb033-Fc), as well as cetuximab and EG2-Fc as a historical control, to inhibit EGF-induced EGFR phosphorylation was tested. At a single concentration of 500 nM, NRC-sdAb032-Fc was the only VHH-Fc showing significant inhibition of EGFR signaling (
Discussion
The VHHs isolated and characterized here were generated by immunization of a llama with DNA alone (no protein or cell boost), using biolistic transfection (gene gun) followed by intradermal injection (DERMOJET). Many of the VHHs had high affinities for recombinant human EGFR, cross-reacted with rhesus and/or mouse EGFR, and recognized native cell-surface EGFR on tumor cell lines; in all of these respects, the VHHs described here are dramatically superior to those previously isolated by our group using recombinant protein immunization [4]. One VHH, NRC-sdAb032, showed clear functional activity as an inhibitor of EGFR signaling, and any of the humanized VHHs may have other therapeutic applications. NRC-sdAb032 was isolated by panning on streptavidin-captured EGFR but not on passively adsorbed EGFR, and its epitope appears to be present on native EGFR and recombinant EGFR ectodomain in solution but not in the same ectodomain when it is passively adsorbed to microtiter plates or amine-coupled to sensor chips. This epitope, which overlaps the cetuximab epitope and the EGF-binding site in EGFR domain Ill, is apparently poorly available for binding by NRC-sdAb032 or cetuximab in ELISAs against directly adsorbed EGFR or in SPR experiments in which these antibodies are flowed over amine-coupled EGFR surfaces; in contrast, both NRC-sdAb032 and cetuximab bound EGFR+ tumor cells with lower EC50s than other VHHs with similar monovalent binding affinities for recombinant EGFR. It appears that the NRC-sdAb032 epitope, though easily destroyed, contributes to this VHH's superior recognition of native EGFR displayed on the tumor cell surface.
Humanization of VHHs
With the aim of using these VHHs in therapeutic applications, the sequences of the four VHHs showing significant binding to EGFR-positive tumor cells (NRC-sdAb022, NRC-sdAb028, NRC-sdAb032 and NRC-sdAb033) were humanized with reference to human IGHV3-30*01 and IGHJ1*01 germline genes. Humanization and testing can be accomplished using routine techniques. This process yielded at least one humanized variant with unimpaired solubility and EGFR-binding affinity for each of three parent VHHs (NRC-sdAb028-H1, NRC-sdAb032-H1 and NRC-sdAb033-H2). The framework regions of these humanized variants bore 89-94% sequence identity with human IGHV3-30*01, with no or minimal impact on the biophysical properties and EGFR-binding affinities of the resulting VHHs (Tables 4 and 5).
1Determined by SEC (% monomer peak area).
2n.d., not determined due to low or no expression.
3n.b., no binding of the purified monomeric sdAb to human EGFR-Fc could be detected.
4n.d., not determined because steady-state KD was calculated.
aIMGT framework definitions
Generation of Chimeric Antigen Receptor Constructs
Overview
Chimeric antigen receptor (CAR) technology has revolutionized the treatment of B-cell malignancies and steady progress is being made towards CAR-immunotherapies for solid tumours. In the context of CARs targeting antigens which are commonly overexpressed in cancer but also expressed at lower levels in normal tissues, such as epidermal growth factor family receptors EGFR or HER2, it is imperative that any targeting strategy consider the potential for on-target off-tumour toxicity. Molecular optimization of the various protein domains of CARs can be used to increase the tumour selectivity.
Herein, high-throughput CAR screening is used to identify a novel camelid single-domain antibody CAR (sdCAR) targeting human epidermal growth factor (EGFR) with high EGFR-specific activity. To further optimize the target selectivity of this EGFR-sdCAR, progressive N-terminal single amino acid truncations of an extended human CD8 hinge domain [(G4S)3GG-45CD8h] were made tested to improve selectivity for EGFR-overexpressing cells. Varying hinge domains in scFv-based CARs targeting EGFR-family tumour associated antigens EGFRvII and HER2 were compared.
Through comparison of various hinge-truncated scFv- and sdAb-based CARs, it is shown that the CAR hinge/spacer domain plays varying roles in modifying CAR signaling depending upon target epitope location. For membrane-proximal epitopes, hinge truncation by even a single amino acid resulted in fine control of CAR signaling strength. Hinge-modified CARs showed consistent and predictable signaling in Jurkat-CAR cells and primary human CAR-T cells in vitro and in vivo. Overall, these results indicate that membrane-proximal epitope targeting CARs can be optimized through hinge length tuning for improved target selectivity and therapeutic function.
Materials and Methods
CAR cloning
Three of the above-described EGFR-specific sdAb sequences were cloned into a modular CAR backbone using PCR amplification and single-pot restriction ligation as previously described[47]. EGFR-sdCAR constructs bearing either a full-length human 45 amino acid CD8 hinge (45CD8h) or progressively N-terminally truncated hinge variants (34CD8h, 22CD8h, or no hinge) were cloned using Gibson assembly. A library of sdAb021-CAR truncation mutants with single amino acid N terminal truncations of the human CD8 hinge extended with an additional N-terminal flexible linker [(GGGGS)3GG-CD8h] was generated using a modular hinge-CAR with convenient type-IIs restriction sites integrated into the construct 3′ of the sdAb coding region. An array of DNA encoding truncated CD8 hinge domains of varying lengths (60 to 1 amino acid) was synthesized as DNA fragments (Twist Bioscience, USA) and cloned into the sdAb021-modular-hinge-BBz-GFP CAR construct using single-pot restriction ligation. Limited hinge truncation libraries with defined-target CARs were generated by exchanging the sdAb021 sequence with appropriate scFv sequences. Trastuzumab derived scFv sequences were generated based on previously reported mutant forms of trastuzumab with enhanced avidity, whereas EGFRvIII-targeting scFvs were generated as previously reported[47]. Both HER2- and EGFRvIII-scFvs were in a VH-(G4S)3-VL format.
Cell Lines and Culture
All cell lines were purchased from American Tissue Culture Collection (ATCC, Manassas, VA). The glioblastoma cell line U97MG-WT and U87MG-vIII (U87-vIII, expressing EGFRvIII via retroviral transduction and sorting) were kindly provided by Professor Cavnee, from the Ludwig Institute for Cancer Research, University of California, San Diego (San Diego, CA, USA). Cell lines used were Jurkat, and target cells SKOV-3, MCF-7, U-87-MG vll, Raji, and Nalm6. Target cells were transduced with lentivirus containing NucLight Red (Sartorius, Essen BioScience, USA), a third generation HIV-based, VSV-G pseudotyped lentivirus encoding a nuclear-localized mKate2. All cell lines were cultured in RPMI supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. These cell lines were tested for the presence of mycoplasma contamination by PCR.
CAR-J Assay
Jurkat cells were transfected via electroporation according to a previously outlined protocol[47]. Briefly, 5×10{circumflex over ( )}5 cells were suspended in 100 μl Buffer 1SM (5 mM KCl, 15 mM MgCl2, 120 mM Na2HPO4/NaH2PO4, 25 mM sodium succinate, and 25 mM mannitol; pH7.2) and incubated with 2 μg of pSLCAR-CAR plasmids as described in the text or with no plasmid control. Cells and plasmid DNA in solution were transferred into 0.2 cm generic electroporation cuvettes (Biorad Gene Pulser; Bio-Rad Laboratories, Hercules, California, USA) and immediately electroporated using a Lonza Nucleofector I (Lonza, Basel, Switzerland) and program X-05 (X-005 on newer Nucleofector models). Cells were cultured in pre-warmed recovery media (RPMI containing 20% FBS, 1 mM sodium pyruvate and 2 mM L-glutamine) for four 4h before being co-cultured with EGFR-expressing target cells U-87MG-vIII, MCF7 and SC-OV-3 or negative control Ramos and Nalm6. Electroporated Jurkat cells were added to varying numbers of target cells in round bottom 96-well plates in effector to target (E:T) ratios ranging from 1:10 to 100:1 (effector to target ratio) or with no target cells (or an E:T of 1:0) and cultured overnight before being staining with allophycocyanin (APC)-conjugated anti human-CD69 antibody (BD Biosciences #555533). Flow cytometry was performed using a BD-Fortessa (BD Biosciences) and data was analyzed using Flowjo software (Flowjo LLC, Ashland, Oregon, USA) and visualized using GraphPad Prism (GraphPad Software, Inc. California, USA).
Human Peripheral Blood Mononuclear Cell (PBMC) Isolation
Heparinized whole blood was collected from healthy donors by venipuncture and transported at room temperature from Ottawa Hospital Research Institute. Blood was diluted 1:1 with Hank's balanced salt solution (HBSS) and PBMCs were isolated by Ficoll density gradient centrifugation. Briefly, samples layered on Ficoll gradient were centrifuged for 20 min at 700 x g without applying a brake. The PBMC interface was carefully removed by pipetting and was washed twice with HBSS by stepwise centrifugation for 15 min at 300 x g. PBMC were resuspended and counted by mixed 1:1 with Cellometer ViaStain™ acridine orange/propidium iodide (AOPI) staining solution and counted using a Nexcelom Cellometer Auto 2000 (Nexcelom BioScience, Lawrence, Massachusetts, USA).
T cells from freshly isolated PBMCs were activated with Miltenyi MACS GMP TransAct CD3/CD28 beads and seeded at 1×106 cells/ml in serum-free StemCell Immunocult-XF media (StemCell Technologies, Vancouver, Canada) containing clinical grade human IL-2 (20 U/ml, Novartis) or Miltenyi TexMACS containing research grade human IL-7 and human IL-15 (both 10 ng/ml).
T cells from freshly isolated PBMC were activated with Miltenyi MACS GMP TransAct CD3/CD28 beads and seeded 1×106 T cells/ml in serum-free StemCell Immunocult-XF media (StemCell Technologies, Vancouver, Canada) with clinical grade 20 U/ml human IL-2 (Novartis) or Miltenyi TexMACS with research grade 10 ng/ml human IL-7 and 10 ng/ml human IL-15.
Human Primary T Transduction by Spinfection
High concentration lentiviral particles encoding various sdCAR constructs were generated as previously described [47]. After 24 h of T cell stimulation with beads, T cells were transduced with sdCAR-GFP lentiviral vectors (multiplicity of infection=10) by spinfection. Briefly, lentivirus was added to T cells (1×106 cells/ml) and the mixture was centrifuged at 850×g for 2 h at 32° C. After centrifugation, cells were incubated at 37° C. for another 2 h. After incubation, cells were plated in a 24 well plate (100,000 cells/ml/well in a total of 1.5 mL) in media supplemented with cytokine(s). Media with cytokine(s) was added at 48 and 72 h post transduction to promote CAR-T cell proliferation without disrupting the cells. CAR-T cells were sampled daily until the end of production. Cell number and viability were assessed by AOPI staining and counting using a Nexcelom Cellometer. CAR-T cells were propagated until harvest on days 7, 9, 14, and 21 to assess the efficiency of transduction and to characterize T cell subpopulations by flow cytometry. CAR-T cells that had returned to a resting state (as determined by decreased growth kinetics, day 10 post-T cell activation) were used for assays. Expression of GFP-CARs by transduced T cells ranged from 20% to 70%.
Continuous Live-Cell Imaging Cytotoxicity Assay
Cytotoxicity of the CAR-T cells was assayed using a Sartorius IncuCyte S3 (Essen Bioscience). Tumour cells (U87vIII-NucLight, MCF7-NucLight, and SKOV3-NucLight were plated in a flat bottom 96-well plate (2000 cells/well). CAR-T cells or control T cells were added into each well in a final volume of 200 μl per well at varying effector:target ratios and co-cultured for 7 days at 37° C. Images were taken every 30 min in light phase and under red (ex. 565-605 nm; em. 625-705 nm) or green fluorescence (ex. 440-480 nm; em. 504-544 nm). The assays were repeated twice with T cells derived from independent blood donors. For one donor, CAR-T cells challenged once or twice with EGFR-high SKOV3 cells were rechallenged with various freshly plated target cells after 7 day of co-culture. Automated cell counting of red (target) or green (CAR-T) cells was performed using Incucyte analysis software and data were graphed using GraphPad Prism.
Animal Studies
NOD/SCID/IL2Ry−/− (NSG, JAX #005557) mice were purchased from Jackson Laboratories and maintained by the Animal Resource Group at the National Research Council of Canada. Eight-week-old NSG mice were injected with 2×106 SKOV-3 in 100 μL of PBS subcutaneously. Eighteen days post tumour cells injection (when tumour reached 5 mm×5 mm), mice were retro orbitally injected with 5×106 mock T cells or T cells transduced with various CAR-T cells as described in the text. Tumours were measured using calipers twice a week and mice were imaged via IVS in vivo imager for red-fluorescence signal (expressed on tumour cells) once a week. For the alternative U87vIII model experiments, mice were subcutaneously injected with 1×106 fluorescently labelled U87-vIII cells described above, a number previously determined to consistently produce a palpable tumour within 7 days. Eight days after tumour cell injection, cryo-preserved CAR-T cells were thawed, washed with PBS, and 1×107 total T cells (with 20-25% CAR transduction) were immediately delivered intra-tumourally, ensuring equal distribution of tumour sizes between groups. Tumour growth was evaluated three times per week using calipers by trained animal technicians blinded to specific treatment groups. Primary endpoint was tumour size above 2000 mm3, with secondary endpoints determined by overall animal health and well-being. Mice were also assessed for tumour growth using IVIS in vivo imaging to examine red fluorescence derived from the NLS-mKate2 marked U87vIII cells. Mice were euthanized when they met pre-specified endpoints. The study was approved by the NRC-HHT Institutional Animal Care Committee and was conducted in accordance with Canadian Council on Animal Care (CCAC) guidelines. Tumour growth and survival (humane endpoint) curves were generated using GraphPad Prism.
Flow Cytometry and Antibodies
Blood was obtained from mice at various time points post CAR-T injection. Blood was washed with cold PBS and pelleted at 350×g for 5 min at 4° C. Red blood cells were lysed using Red Blood Cell Lysing Buffer Hybri-Max (Sigma-Aldrich, St. Louis, MO, USA). Human T cells were identified and analyzed for activation/differentiation status using the following antibodies: hCD45-APC-H7, hCD45RA-BV650, hCD45RO-PE-CF594, hCD27-BUV737, hCCR7-PE, hCD4-BUV395, and hCD8-PerCP-Cy5.5 (all antibodies from BD Biosciences, USA). CAR expression was measured indirectly via expression of GFP incorporated in CAR constructs. To evaluate exhaustion, staining by an hPD-1-BV421 antibody was evaluated. T cell activation was detected using hCD25-PE-Cy7 and hCD69-BV786 antibodies. For in vivo studies, a BV71 1-labeled antibody against mouse CD45 was used to identify murine cells and CD19 expression was analyzed using an anti-human CD19-BUV496 antibody. Staining of human EGFR was performed using anti-human EGFR-PE-CF594 (BD Biosciences, Cat #563431).
Results
High-Affinity EGFR-Specific sdAbs Generate Antigen-Responsive CARs with Low Tonic Signaling
Camelid sdAbs were raised in a previous study against EGFR using DNA immunization and phage display. In order to assess whether these sdAbs were functional in the context of a sdCAR three EGFR-specific sdAbs with varying affinities as discussed above and epitopes were selected (Table 6).
The sdAbs were cloned into a modular CAR backbone and the resulting sdCARs were screened for responses to target cells with varying EGFR expression using a previously described high throughput Jurkat screening assay[47] (
Hinge Shortening Decreases CAR Signaling
It has previously been shown that altering the length of the spacer (hinge) between the antigen binding domain (ABD) and transmembrane domain can be used to modulate CAR signaling[48,49]. It was next investigated whether the selectivity of the EGFR sdCARs for EGFR-high cells could be increased by progressively decreasing the length of the hinge region. EGFR sdCAR constructs were cloned with either a full-length 45 amino acid human CD8 hinge (45CD8h) or progressively N-terminally truncated hinge variants (34CD8h, 22CD8h, or no hinge) (
Experiments were designed to more finely map the effects of hinge length modulation on an EGFR sdAb CAR. As a starting point, an extended hinge domain was designed wherein an additional N-terminal flexible linker of 17 amino acids was included before the human CD8-hinge sequence [(GGGGS)3GG-CD8h]. A library of sdAb021-CAR constructs with single amino acid N-terminal deletions of the human CD8 hinge was generated. Screening the sdCAR single-residue hinge truncation library revealed a clear pattern of CAR activation (
Examining the range of CAR activity across the full array of single-amino acid truncations tested, it is expected that particular ranges of hinge length may have applicability to different cellular immunotherapy targeting strategies (
Epitope Location is a Determinant of CAR Hinge Sensitivity
As has been previously proposed, the location of the CAR target epitope should be critical in determining the minimal hinge length necessary for CAR signaling[50]. Epitope binning experiments indicated that all three sdAbs have partially overlapping epitopes, and cross-reactivity analysis is suggestive of membrane proximal domain IV binding (Table 6). To more carefully investigate the role of epitope location, limited hinge libraries were generated for CARs with known target epitopes. scFv-CARs were generated based on either trastuzumab, which is known to bind a highly membrane proximal epitope of HER2 [51], or novel EGFRvII-specific antibodies[47], which by necessity must bind the membrane distal neo-epitope of EGFRvII (Table 6). As hypothesized, the trastuzumab CAR required a very long hinge element: no HER2 scFv CARs containing shorter than a full CD8-hinge showed any response (
Hinge-Truncated CAR Signaling is Consistent in Primary T Cells In Vitro and In Vivo
Experiments were carried out to confirm whether the reduced signaling of sdCARs with truncated hinge elements expressed in Jurkat cells was also manifested in primary CAR-T cells. Thus, T cells derived from two human blood donors were transduced with hinge-modified forms of the sdAb021 sdCAR. Following polyclonal expansion and sdCAR transduction, green fluorescent protein (GFP)+ CAR-T cells with various hinge elements were placed in low density co-culture with target cells stably marked with a nuclear localized red fluorescent protein. Co-cultures were then monitored for tumour cell (red fluorescence) and CAR-T cell (green fluorescence) expansion over 7 days. Consistent with observations in Jurkat cells, hinge truncation progressively diminished the ability of sdAb021 sdCARs to restrict tumour cell growth and expand in response to target cells (
Experiments were carried out to more finely examine the effect of hinge truncation under a wider variety of antigenic conditions. Thus, hinge-modified sdAb021 sdCAR-T cells were plated with EGFR-very high (SKOV3), EGFR-medium/high (U87vIII), or EGFR-low (MCF7) target cells at varying effector:target ratios and examined them using live microscopy. Using primary T cells from both donors, sdCAR-T cells with truncated hinges maintained tumour killing and CAR-T expansion in response to EGFR-overexpressing SKOV3 and U87vIII cells but showed low responses to EGFR-low MCF7 cells (
The activity of hinge-truncated CAR molecules against primary human skin fibroblast cells was also tested, which show a relatively high level of EGFR expression as measured using flow cytometry and a commercial anti-EGFR antibody (
Hinge-Truncated CARs Maintain Selectivity for High-Expressing Cells Following Re-Challenge
The effect of hinge truncation on serial CAR-T killing was investigated. Hinge modified sdCAR-T cells were isolated following primary challenge with antigen-overexpressing SKOV3 cells using the low density co-culture assay as described above and re-challenged the sdCAR-T cells by re-plating with additional target cells (
To specifically address the question of whether sdCARs with truncated hinges maintained their selectivity following antigen experience, single or double SKOV3-challenged sdCAR-T cells were re-challenged with U87vIII (
In Vivo CAR-T Response is Progressively Diminished with Hinge Truncation
Experiments were carried out to investigate whether in vitro observations regarding the relationship between hinge length and CAR-T activity were consistent in an in vivo xenograft model. Using the relatively slow growing SKOV3 model, mice were challenged with 2 million tumour cells subcutaneously and then treated intravenously at day 18 post-tumour implantation with 5 million hinge modified sdCAR-T cells or 1.25-1.5 million mock transduced CAR-T cells. There was a progressive increase in tumour growth in all mice (
Examining CAR-T cells in the blood of treated mice revealed a consistent pattern of increased expansion of sdCAR-T cells with longer hinge regions at 43 and 54 days post-tumour injection (
Inclusion of a Hinge Element can Increase Bi-Specific T-Cell Engaging Antibody Activity
It was next assessed whether inclusion of a human CD8-hinge domain similar to that found in the CAR molecules discussed above can also increase the activity of a bi-specific antibody which can simultaneously engage both a human T-cell through the CD3 receptor and a human EGFR-expressing target cell. Thus a bi-specific antibody composed of a human CD3-specific single-chain variable fragment and an EGFR-specific sdAb021 camelid single domain antibody fragment, linked either by a short GGGGS linker alone or a long human (G4S)3-45CD8-hinge-G4S linker, were generated (
Discussion
Novel CAR constructs were sought that would be able to effectively target cells overexpressing EGFR and to discriminate between high level expression on tumours and lower expression on normal cells. Despite some variation in binding affinity for the three EGFR sdAb moieties tested here (Table 6), all three sdCAR constructs showed strong responses to EGFR-high SKOV3 and EGFR-low MCF7 target cells. Intriguingly, all EGFR-sdAb CAR receptors showed responsiveness to MCF7 cells despite no apparent reactivity of the purified sdAbs to EGFR-low MCF7 cells. These results are consistent with previous observations that EGFR specific CARs are relatively insensitive to ABD affinity up to the micromolar range[46] and underscore the exquisite antigen sensitivity of CAR-T cells to respond to and lyse even very low antigen expressing target cells[52]. This phenomenon possibly relates to the extreme multi-valency of both CAR and EGFR on their respective cells. Increased valency is well known to lead to significant avidity effects that boost the apparent affinity of biological interactions[53], and these would presumably apply to CAR-T cells as well.
While CAR constructs with maximal antigen sensitivity might be desirable in certain contexts, such as for CAR-T therapies targeting B cell family restricted antigens, the presence of EGFR expression on normal tissue requires a targeting strategy that would be selective for EGFR-overexpressing cancer cells. Previously, affinity modulation has been used to increase selectivity of CAR constructs for overexpressing cells[46]. While it is certainly possible to decrease sdAb affinity through various molecular strategies[54], mutational antibody changes can sometimes lead to unpredictable binding behaviour such as unexpected off-target binding, elevated tonic signaling, or loss of efficacy. Thus, an alternate strategy was pursued to decrease on target activity through hinge modification.
It is shown herein that even very small truncations of a human CD8 hinge, down to the level of individual amino acids, can be a powerful and remarkably precise tuning mechanism for CAR signaling. For the EGFR sdCAR tested most extensively, there was a sharp drop-off of CAR signaling over a range of only 10 amino acids within the CD8 hinge motif. While the requirements of lentiviral production and primary T cell transduction dictated that only a limited number of constructs in primary T cells could be tested, it would be intriguing to map the optimal hinge length for antigen induced CAR-T killing or CAR-T expansion in genuine donor-derived T cells. The hinge-truncation data using either membrane proximal (trastuzumab) or membrane-distal (anti-EGFRvIII-mAbs) based CAR constructs provides a clear demonstration of the criticality of epitope location as a determinant of hinge-sensitivity for CAR molecules reacting to tumour cells.
While the exact epitope for the EGFR sdAbs tested is not known, the data here would indicate a likely membrane proximal location. Consistent with this, sdAb028 cross-reacts with human and mouse but not cynomolgus EGFR; the only positions at which mouse and human EGFR sequences are identical but diverge from cynomolgus EGFR are in domain IV of EGFR. The finding that a full length CD8 hinge is required for the trastuzumab scFv-CAR seems to be consistent with previous experiments using similar CARs where hinge domains were also included[57], although it is worth noting that the scFv employed there diverges somewhat from that used here. In contrast to hinge truncation, it was found that a longer than necessary hinge does not have a very deleterious effect on CAR signaling, at least as determined by the CAR-J assay. Previous work has indicated that longer hinges can decrease in vivo activity for membrane-distal epitope targeting CARs[55], but follow-up studies pinpointed the effect to be related to FC-binding by IgG-hinge motifs rather than hinge length specifically[56]. For those membrane-proximal targeting CARs where a hinge is required though, the data demonstrates that CD8-hinge truncation is a powerful molecular strategy to reduce CAR-antigen sensitivity without the need to alter the ABD.
Due to the relatively more demanding technical requirements of testing CAR-T constructs in primary T cells only a limited number of CAR constructs was tested. Nonetheless, data presented here provide additional evidence that molecular optimization using transient CAR expression in Jurkat cells is predictive of signaling in stably transduced primary CAR-T cells, as has been previously reported[47]. The wider use of such optimization methodology could lead to improved ABD/hinge design for future CAR products. It may be possible for instance to design CARs with customized signaling for application in CD4, CD8, gamma-delta T cells, or NK cells.
The in vivo experiments presented here underline the fundamental trade-off between on-target, on-tumour activity and strategies that might reduce on-target off-tumour signaling. Achieving a level of signaling that is adequate for potent tumour killing yet also eliminates the risk of on-target toxicity may be difficult. Although in isolation, the SKOV3-selective truncated EGFR-sdAb CARs which were tested here had relatively little therapeutic effect in vivo it may be possible to combine such reduced signal hinge-truncated EGFR-sdCAR in a multi-antigen targeting CAR strategy that will ultimately more effectively recognize and lyse tumour cells in a highly selective fashion. Importantly, the aggressive xenograft models used here are imperfect and would likely not perfectly predict CAR-T signaling and activity in humans with slower growing and/or metastatic disease. The clinical use of CARs with varying hinge lengths could also present an intriguing alternative safety pathway for clinical trials of solid tumour targeting CARs through hinge length escalation.
In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the embodiments. However, it will be apparent to one skilled in the art that these specific details are not required.
The above-described embodiments are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.
The following publications are incorporated by reference herein.
This application claims the benefit of priority from U.S. Provisional Application No. 63/167,413 which is entitled “ANTI-EGFR SINGLE DOMAIN ANTIBODIES AND THERAPEUTIC CONSTRUCTS” and was filed Mar. 29, 2021, which is incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2021/051525 | 10/28/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63167413 | Mar 2021 | US |
