NUCLEIC ACIDS ENCODING A POLYPEPTIDE COMPRISING A MODIFIED FC REGION OF A HUMAN IGG1 AND AT LEAST ONE HETEROLOGOUS ANTIGEN
20230310584 · 2023-10-05
Inventors
- Linda Gillian Durrant (Oxford Oxfordshire, GB)
- Mireille Vankemmelbeke (Oxford Oxfordshire, GB)
- Victoria Anne Brentville (Oxford Oxfordshire, GB)
- Rachael Louise Metheringham (Oxford Oxfordshire, GB)
Cpc classification
A61K39/001156
HUMAN NECESSITIES
C07K2319/30
CHEMISTRY; METALLURGY
C07K2319/40
CHEMISTRY; METALLURGY
C12N2770/20034
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
A61K39/215
HUMAN NECESSITIES
C07K16/283
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K14/4748
CHEMISTRY; METALLURGY
C07K2317/34
CHEMISTRY; METALLURGY
C12N2770/20022
CHEMISTRY; METALLURGY
International classification
A61K39/215
HUMAN NECESSITIES
C07K16/28
CHEMISTRY; METALLURGY
A61K39/00
HUMAN NECESSITIES
Abstract
The present invention relates to nucleic acids and peptides encoded by those nucleic acids. In particular, the peptides comprise a modified IgG1 Fc region and one or more heterologous epitopes, which may be B- or T-cell epitopes. A nucleic acid of the invention may encode a polypeptide comprising: (i) a modified Fc region of a human IgG1, and (i) at least one heterologous antigen, wherein (a) the modified Fc region comprises at least the part of Fc that is capable of binding to CD64 and/or TRIM21, (b) at least one residue of the Fc region is modified to the corresponding residue from a mouse IgG3 antibody and (c) the modified Fc region has enhanced avidity for Fc-gamma receptor (FcγR) when compared to the corresponding wildtype Fc region.
Claims
1-2. (canceled)
3. A nucleic acid which encodes a polypeptide comprising: (i) a modified Fc region of a human IgG1, and (ii) at least one heterologous antigen, wherein (a) the modified Fc region comprises at least the part of Fc that is capable of binding to CD64 and/or TRIM21, (b) at least one residue of the Fc region is modified to the corresponding residue from a mouse IgG3 antibody and (c) the modified Fc region has enhanced avidity for Fc-gamma receptor (FcγR) when compared to the corresponding wildtype Fc region.
4. (canceled)
5. The nucleic acid of claim 1, wherein the at least one residue of the Fc region is selected from: N286, K288, K290, A339, Q342, P343, R344, E345, L351, S354, D356, E357, L358, T359, N361, Q362, K370, G371, Y373, P374, S375, D376, A378, optionally wherein the at least one modified residue is selected from: N286T, K288W, K290Q, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.
6. The nucleic acid of claim 5, wherein the Fc region of human IgG3 comprises all of the following modifications: N286T, K288W, K290Q, A339P, Q342R, P343A, R344Q, E345T, L351I, S354P, D356E, E357Q, L358M, T359S, N361K, Q362K, K370T, G371N, Y373F, P374S, S375E, D376A, A378S.
7. The nucleic acid of claim 6, wherein the modified Fc region comprises the amino acid sequence provided in SEQ ID NO: 1, or an amino acid sequence having at least 90% identity to SEQ ID NO: 1.
8. The nucleic acid of claim 1, wherein the at least one heterologous antigen is linked, directly or via a linker, to the N-terminus of the modified human IgG1 Fc region.
9. The nucleic acid of claim 1, wherein the polypeptide further comprises an antibody variable region into which the at least one heterologous antigen is inserted or substituted, optionally wherein the at least one heterologous antigen is substituted into a CDR of the antibody variable region.
10. The nucleic acid of claim 1, wherein the at least one heterologous antigen comprises a T cell epitope and/or a B cell epitope, and/or the at least one heterologous antigen is from a cancer or an infectious disease, and/or the at least one heterologous antigen comprises one or more epitopes selected from the epitopes set out in any one of
11-13. (canceled)
14. The nucleic acid of claim 1, wherein the at least one heterologous antigen comprises one or more epitopes selected from: TABLE-US-00017 (a) (SEQ ID NO: 29) GTGRAMLGTHTMEVTVYH; (b) (SEQ ID NO: 30) SVYDFFVWL; (c) (SEQ ID NO: 31) WNRQLYPEWTEAQRLD, or one or more epitopes selected from: TABLE-US-00018 (a) (SEQ ID NO: 29) GTGRAMLGTHTMEVTVYH; (b) (SEQ ID NO: 30) SVYDFFVWL; (c) (SEQ ID NO: 31) WNRQLYPEWTEAQRLD; and (d) (SEQ ID NO: 32) VPLDCVLYRYGSFSVTLDIVQG, or one or more epitopes selected from: TABLE-US-00019 (a) (SEQ ID NO: 29) GTGRAMLGTHTMEVTVYH; (b) (SEQ ID NO: 30) SVYDFFVWL; (c) (SEQ ID NO: 31) WNRQLYPEWTEAQRLD; (d) (SEQ ID NO: 32) VPLDCVLYRYGSFSVTLDIVQG; (e) (SEQ ID NO: 33) ANCSVYDFFVWLHYYSVRDTLLGPGRPYR; and (f) (SEQ ID NO: 34) QCTEVRADTRPWSGPYILRNQDDRELWPRKFF.
15-16. (canceled)
17. The nucleic acid of claim 1, wherein the at least one heterologous antigen comprises one or more epitopes selected from: TABLE-US-00020 (a) (SEQ ID NO: 35) LLMWITQCF; (b) (SEQ ID NO: 36) SLLMWITQC; (c) (SEQ ID NO: 37) PESRLLEFYLAMPFATPMEAELARRSLAQ; and (d) (SEQ ID NO: 38) PGVLLKEFTVSGNILTIRLTAADHR, or one or more epitopes selected from: TABLE-US-00021 (a) (SEQ ID NO: 35) LLMWITQCF; (b) (SEQ ID NO: 36) SLLMWITQC; (c) (SEQ ID NO: 37) PESRLLEFYLAMPFATPMEAELARRSLAQ; (d) (SEQ ID NO: 39) PESRLLEFY; (e) (SEQ ID NO: 40) RLLEFYLAMPFATP; (f) (SEQ ID NO: 41) LEFYLAMPF; (g) (SEQ ID NO: 42) EFYLAMPFATPM; (h) (SEQ ID NO: 43) MPFATPMEA; (i) (SEQ ID NO: 44) LAMPFATPM; (j) (SEQ ID NO: 45) LLEFYLAMPFATPM; (k) (SEQ ID NO: 46) LLEFYLAMPFATPMEAELARRSLAQ; (l) (SEQ ID NO: 38) PGVLLKEFTVSGNILTIRLTAADHR; (m) (SEQ ID NO: 47) LKEFTVSGNILTIRL; (n) (SEQ ID NO: 48) KEFTVSGNILT; (o) (SEQ ID NO: 49) KEFTVSGNILTI; (p) (SEQ ID NO: 50) TVSGNILTIR; and (q) (SEQ ID NO: 51) TVSGNILTI.
18. (canceled)
19. (canceled)
20. The nucleic acid of claim 9, wherein the antibody variable region is a heavy chain variable region comprising the following heterologous antigens substituted into the CDR1, CDR2 and CDR3 respectively: TABLE-US-00022 (a) (SEQ ID NO: 29) GTGRAMLGTHTMEVTVYH, (SEQ ID NO: 30) SVYDFFVWL and (SEQ ID NO: 32) VPLDCVLYRYGSFSVTLDIVQG; or (b) (SEQ ID NO: 35) LLMWITQCF, (SEQ ID NO: 36) SLLMWITQC and (SEQ ID NO: 37) PESRLLEFYLAMPFATPMEAELARRSLAQ.
21-27. (canceled)
28. The nucleic acid of claim 1, in combination with a second nucleic acid encoding at least one heterologous antigen.
29-34. (canceled)
35. The nucleic acid of claim 28, wherein the second nucleic acid encodes an antibody light chain into which the at least one heterologous antigen is inserted or substituted, optionally wherein the at least one heterologous antigen is substituted into a CDR of the antibody light chain.
36. The nucleic acid of claim 35, wherein the antibody light chain encoded by the second nucleic acid comprises the following heterologous antigens substituted into the CDR1, CDR2 and CDR3 respectively: WNRQLYPEWTEAQRLD (SEQ ID NO: 31), ANCSVYDFFVWLHYYSVRDTLLGPGRPYR (SEQ ID NO: 33) and QCTEVRADTRPWSGPYILRNQDDRELWPRKFF (SEQ ID NO: 34) or the antibody light chain encoded by the second nucleic acid comprises the sequence PGVLLKEFTVSGNILTIRLTAADHR (SEQ ID NO: 38) substituted into the CDR2, or the antibody light chain encoded by the second nucleic acid comprises the amino acid sequence provided in SEQ ID NO: 10 or SEQ ID NO: 11, or the polypeptide encoded by the nucleic acid comprises the amino acid sequence provided in SEQ ID NO: 2 and wherein the antibody light chain encoded by the second nucleic acid comprises the amino acid sequence provided in SEQ ID NO: 10, or the polypeptide encoded by the nucleic acid comprises the amino acid sequence provided in SEQ ID NO: 3 and wherein the antibody light chain encoded by the second nucleic acid comprises the amino acid sequence provided in SEQ ID NO: 11.
37-40. (canceled)
41. A vector comprising the nucleic acid of claim 1.
42. The vector of claim 41, wherein the vector further comprises a second nucleic acid encoding at least one heterologous antigen, and/or the vector is a DNA plasmid or doggybone (dbDNA) vector, and/or the vector comprises the nucleotide sequences of: (a) SEQ ID NO: 12 and SEQ ID NO: 13; or (b) SEQ ID NO: 14 and SEQ ID NO: 15; or (c) SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.
43-49. (canceled)
50. A peptide encoded by the nucleic acid of claim 1.
51. A vaccine composition comprising the nucleic acid of claim 1, optionally in combination with an adjuvant.
52. (canceled)
53. A method of preventing or treating cancer in a subject in need thereof, the method comprising administering the vaccine composition of claim 51.
54. (canceled)
55. The method of claim 53, wherein the vaccine composition is administered to the subject using needle-free injection, and/or wherein two or more of the vaccine compositions are administered to the subject, wherein the two or more vaccine compositions are different.
56-57. (canceled)
58. The method of claim 53, wherein the cancer is melanoma.
59. A vaccine composition comprising the vector of claim 41, optionally in combination with an adjuvant.
60. A vaccine composition comprising the peptide of claim 50, optionally in combination with an adjuvant.
61. A method of preventing or treating cancer in a subject in need thereof, the method comprising administering the vaccine of claim 59.
62. A method of preventing or treating cancer in a subject in need thereof, the method comprising administering the vaccine of claim 60.
Description
EXAMPLES
[0210] The present invention will now be described further with reference to the following examples and the accompanying drawings. Future data collection from healthy volunteers immunised, using electroporation, with the novel pDNA vaccine, engineered to induce both VNAbs and potent T cells have been informed as a direct result of the example outcomes.
[0211]
Depicting cloning sites BamHI*/XhoI* and HindIII**/PstI** utilised for excision of light and heavy chain and replacement with S and N sections in first* and second** round of cloning respectively.
[0212]
Nucleotide and amino acid sequence of the S glycoprotein and N full length chains within the expression vector pVAXDC. The S chain encodes RBD amino acids 319-541 and a murine IgK leader. The nucleoprotein chain encodes amino acids 2-419 fused inframe with the human IgG1 hinge-CH2-CH3 along with the murine IgK leader. The stop codon is depicted by an asterix. The BamHI/XhoI and HindIII/PstI restriction sites utilised in transfer of both chains are highlighted.
[0213]
Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC. Both chains contain a murine IgK leader. The S chain encodes RBD amino acids 319-541 linked via a glycine serine to a fibritin trimer motif. The N chain encodes amino acids 2-419 fused inframe with the human igG1 hinge-CH2-CH3. The stop codon is depicted by an asterix. The BamHI/XhoI and HindIII/PstI restriction sites utilised in transfer of both chains are highlighted.
[0214]
Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC. Both chains contain a murine IgK leader. The S chain encodes RBD amino acids 319-541 fused inframe with the human IgG1 hinge-CH2-CH3. The N chain encodes amino acids 2-419. The stop codon is depicted by an asterix. The BamHI/XhoI and HindIII/PstI restriction sites utilised in transfer of both chains are highlighted.
[0215]
Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC. Both chains contain a murine IgK leader. The S glycoprotein chain encodes RBD amino acids 319-541 while the N chain encodes amino acids 2-419. The stop codon is depicted by an asterix. The BamHI/XhoI and HindIII/PstI restriction sites utilised in transfer of both chains are highlighted.
[0216]
Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC. The S chain encodes RBD amino acids 319-541 and a human IgH leader. The N chain encodes amino acids 2-419 fused in frame with the human IgG1 hinge-CH2-CH3 along with the human IgH leader. The stop codon is depicted by an asterix. The BamHI/XhoI and HindIII/PstI restriction sites utilised in transfer of both chains are highlighted.
[0217]
Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC. Both chains contain a human IgH leader. The S chain encodes RBD amino acids 319-541 linked via a glycine serine to a fibritin trimer motif. The N chain encodes amino acids 2-419 fused in frame with the human IgG1 hinge-CH2-CH3. The stop codon is depicted by an asterix. The BamHI/XhoI and HindIII/PstI restriction sites utilised in transfer of both chains are highlighted.
[0218]
Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC. Both chains contain a human IgH leader. The S chain encodes RBD amino acids 319-541 fused inframe with the human IgG1 hinge-CH2-CH3. The N chain encodes amino acids 2-419. The stop codon is depicted by an asterix. The BamHI/XhoI and HindIII/PstI restriction sites utilised in transfer of both chains are highlighted.
[0219]
Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC. Both chains contain a human IgH leader. The S glycoprotein chain encodes RBD amino acids 319-541 while the N chain encodes amino acids 2-419. The stop codon is depicted by an asterix. The BamHI/XhoI and HindIII/PstI restriction sites utilised in transfer of both chains are highlighted.
[0220]
Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC. Both chains contain a human IgH leader. The S chain encodes RBD amino acids 330-525 attached via a longer (GGGS).sub.3GS glycine serine linker to a fibritin trimer motif (GTGGGSG). The N chain encodes amino acids 2-419 fused in frame with the human IgG1 hinge-CH2-CH3. The stop codon is depicted by an asterix. The BamHI/XhoI and HindIII/PstI restriction sites utilised in transfer of both chains are highlighted.
[0221]
Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC. Both chains contain a human IgH leader. The S chain encodes RBD amino acids 330-525 attached via a (GGGS).sub.3 glycine serine linker to a disulphide bridge motif. The N chain encodes amino acids 2-419 fused in frame with the human IgG1 hinge-CH2-CH3. The stop codon is depicted by an asterix. The BamHI/XhoI and HindIII/PstI restriction sites utilised in transfer of both chains are highlighted.
[0222]
Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC. Both chains contain a human IgH leader. The S chain encodes RBD amino acids 330-525 attached via a longer (GGGS).sub.3GS glycine serine linker to a fibritin trimer motif (GTGGGSG). The N chain encodes amino acids 2-419 fused in frame with the human IgG1 hinge-CH2-CH3 iV1 where murine IgG3 23 AA substitutions are highlighted in bold. The stop codon is depicted by an asterix. The BamHI/XhoI and HindIII/PstI restriction sites utilised in transfer of both chains are highlighted.
[0223]
Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC. Both chains contain a human IgH leader. The S chain encodes RBD amino acids 319-541 linked via a glycine serine to a fibritin trimer motif. The N chain encodes amino acids 2-419 fused in frame with the human IgG1 hinge-CH2-CH3 iV1 where murine igG3 23 AA substitutions are highlighted in bold. The stop codon is depicted by an asterix. The BamHI/XhoI and HindIII/PstI restriction sites utilised in transfer of both chains are highlighted.
[0224]
Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC. Both chains contain a human IgH leader. The S chain encodes RBD amino acids 319-541 fused in frame with the human IgG1 hinge-CH2-CH3. The N chain encodes amino acids 2-419 fused in frame with the human IgG1 hinge-CH2-CH3 iV1 where murine igG3 23 AA substitutions are highlighted in bold. The stop codon is depicted by an asterix. The BamHI/XhoI and HindIII/PstI restriction sites utilised in transfer of both chains are highlighted.
[0225]
Nucleotide and amino acid sequence of the S and N full length chains within the expression vector pVAXDC. Both chains contain a human IgH leader. The S chain encodes RBD amino acids 319-541 while the N chain encodes amino acids 2-419 both fused in frame with the human IgG1 hinge-CH2-CH3 iV1 constant region where murine igG3 23 AA substitutions are highlighted in bold. To reduce homology in SN14 between the two codon-optimised human IgG1 hinge-CH2-CH3 iV1 constant regions the nucleotide sequences are not identical. The stop codon is depicted by an asterix. The BamHI/XhoI and HindIII/PstI restriction sites utilised in transfer of both chains are highlighted.
[0226]
[0227]
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[0230]
[0231]
[0232]
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[0238]
Nucleotide and amino acid sequence of the antibody heavy and light variable regions cloned inframe with the human igG1 CH1-hinge-CH2-CH3 constant region and human kappa constant region respectively within the expression vector pVAXDC. Amino acids within boxes encodes the HLA-DR7, HLA-DR53 and HLA-DQ6 restricted gp100.sub.173-190CD4 epitope (GTGRAMLGTHTMEVTVYH) in H1 and L3, the HLA-0201 TRP2.sub.180-188 epitope (SVYDFFVWL) in H2 and the HLA-DR4 restricted gp100.sub.44-59 CD4 epitope in H3 and L1 (WNRQLYPEWTEAQRLD). The HindIII/Afe I and BamHI/BsiWI restriction sites utilised in transfer of the variable heavy and light regions are highlighted. For direct replacement of human igG1 constant domain (CH1-hinge-CH2-CH3) with the enhanced human igG1 CH1-hinge-CH2-CH3 iV1 constant region AfeI and EcoRI were utilised as shown. The stop codon is depicted by an asterisk.
[0239]
[0240]
Nucleotide and amino acid sequence of the antibody heavy and light variable regions cloned inframe with the human igG1 CH1-hinge-CH2-CH3 constant region and human kappa constant region respectively within the expression vector pVAXDC. Amino acids within boxes represent the HLA-DR7, HLA-DR53 and HLA-DQ6 restricted gp100.sub.173-190 CD4 epitope (GTGRAMLGTHTMEVTVYH) in CDR H1, the HLA-0201TRP2.sub.180-188 epitope (SVYDFFVWL) in H2 and the HLA-DR4 restricted gp100.sub.44-59 CD4 epitope in L1 (WNRQLYPEWTEAQRLD) retained from pVAXDCIB68. Additional epitopes include nested within the gp100.sub.471-492 sequence inserted into the H3 site (VPLDCVLYRYGSFSVTLDIVQG) a HLA-A1, B35 and predicted HLA-DP4 epitope. TRP2.sub.177-205 and TRP2.sub.60-91 sequences were grafted into the L2 and L3 sites of the variable light region respectively. These collectively contained an HLA-A2, A3, A31, A33, B35, B44, HLA-DR3 and another potential HLA-DP4 epitope as described elsewhere. The HindIII/Afe I and BamHI/BsiWI restriction sites utilised in transfer of the variable heavy and light regions are highlighted. For direct replacement of human igG1 constant domain (CH1-hinge-CH2-CH3) with the enhanced human igG1 CH1-hinge-CH2-CH3 iV1 constant region AfeI and EcoRI were utilised as shown. The stop codon is depicted by an asterisk.
[0241]
Nucleotide and amino acid sequence of the antibody heavy and light variable regions cloned in frame with the enhanced human IgG1 CH1-hinge-CH2-CH3 iV1 constant region (where murine IgG3 23 AA substitutions are highlighted in bold) and human kappa constant region respectively within the expression vector pVAXDC. Amino acids within boxes represent the HLA-DR7, HLA-DR53 and HLA-DQ6 restricted gp100.sub.173-190 CD4 epitope (GTGRAMLGTHTMEVTVYH) in CDR H1, the HLA-0201 TRP2.sub.180-188 epitope (SVYDFFVWL) in H2 and the HLA-DR4 restricted gp100.sub.44-59 CD4 epitope in L1 (WNRQLYPEWTEAQRLD) retained from pVAXDCIB68. Additional epitopes include, nested within the gp100.sub.471-492 sequence inserted into the H3 site (VPLDCVLYRYGSFSVTLDIVQG), a HLA-A1, B35 and predicted HLA-DP4 epitope. TRP2.sub.177-205 and TRP2.sub.60-91 sequences were grafted into the L2 and L3 sites of the variable light region respectively. These collectively contained an HLA-A2, A3, A31, A33, B35, B44, HLA-DR3 and another potential HLA-DP4 epitope. The HindIII/Afe I and BamHI/BsiWI restriction sites utilised in transfer of the variable heavy and light regions are highlighted. For direct replacement of human IgG1 constant domain (CH1-hinge-CH2-CH3) with the enhanced human IgG1 CH1-hinge-CH2-CH3 iV1 constant region AfeI and EcoRI where utilised as shown. The stop codon is depicted by an asterisk.
[0242]
Nucleotide and amino acid sequence of the antibody heavy and light variable regions cloned inframe with the human igG1 CH1-hinge-CH2-CH3 constant region and human kappa constant region respectively within the expression vector pVAXDC. Amino acids within boxes represent the NYESO-1.sub.158-166 HLA-A24 epitope (LLMWITQCF) and NYESO-1.sub.157-165 HLA-A2-restricted epitope (SLLMWITQC) in CDR H1 and H2. NY-ESO-1.sub.83-111 amino acid sequence (PESRLLEFYLAMPFATPMEAELARRSLAQ) and NY-ESO-1.sub.119-143 (PGVLLKEFTVSGNILTIRLTAADHR) where grafted into the CDR H3 and L1 site which collectively contains a number of nested additional epitopes as described elsewhere (xue et al. ONCOIMMUNOLOGY 2016, VOL. 5, NO. 6, e1169353). The HindIII/Afe I and BamHI/BsiWI restriction sites utilised in transfer of the variable heavy and light regions are highlighted. For direct replacement of human igG1 constant domain (CH1-hinge-CH2-CH3) with the enhanced human igG1 CH1-hinge-CH2-CH3 iV1 constant region AfeI and EcoRI were utilised as shown. The stop codon is depicted by an asterisk.
[0243]
Nucleotide and amino acid sequence of the antibody heavy and light variable regions cloned in frame with the enhanced human IgG1 CH1-hinge-CH2-CH3 iV1 constant region (where murine IgG3 23 AA substitutions are highlighted in bold) and human kappa constant region respectively within the expression vector pVAXDC. Amino acids within boxes represent the NYESO-1.sub.158-166 HLA-A24 epitope (LLMWITQCF) and NYESO-1.sub.157-165 HLA-A2-restricted epitope (SLLMWITQC) in CDR H1 and H2. NY-ESO-1.sub.83-111 amino acid sequence (PESRLLEFYLAMPFATPMEAELARRSLAQ) and NY-ESO-1.sub.119-143 (PGVLLKEFTVSGNILTIRLTAADHR) where grafted into the CDR H3 and L1 site which collectively contains a number of nested additional epitopes as described elsewhere [183]. The HindIII/Afe I and BamHI/BsiWI restriction sites utilised in transfer of the variable heavy and light regions are highlighted. For direct replacement of human IgG1 constant domain (CH1-hinge-CH2-CH3) with the enhanced human IgG1 CH1-hinge-CH2-CH3 iV1 constant region AfeI and EcoRI where utilised as shown. The stop codon is depicted by an asterisk.
[0244]
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[0250]
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[0257]
Nucleotide and amino acid sequence of the spike and nucleoprotein full length chains within the expression vector pVAXDC. Both chains contain a human IgH leader. The spike chain encodes amino acids 319-541 carrying the N501Y mutation of the Kent variant/lineage B.1.1.7, UK-VOC 202012/01. The nucleoprotein chain encodes amino acids 2-419, which includes the D3L and S235F mutations from the variant, fused inframe with the human igG1 hinge-CH2-CH3 iV1 where murine igG3 23 AA substitutions are in bold. The stop codon is depicted by an asterisk. Mutations of the Kent variant/lineage B.1.1.7, UK-VOC 202012/01 are in bold and highlighted in grey. The BamHI/XhoI and HindIII/PstI restriction sites utilised in transfer of both chains are highlighted
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Methods
Materials, Animals, Cells and Antibodies
Peptides and Proteins
[0270] Covid19 peptides were selected based on IEDB database (http://www.iedb.org/) binding predictions for HLA-A*0201, HLA-DR*0101 and HLA-DP*0401 and SYFPEITHI (http://www.syfpeithi.de) binding predictions for HLA-A*0201. Cancer antigen peptides were selected based on published sequences, IEDB database (http://www.iedb.org/) binding predictions and SYFPEITHI (http://www.syfpeithi.de) binding predictions. Peptides (Table 2) were synthesized at >90% purity (Genscript), aliquoted to single use vials and stored lyophilized at −80° C. then reconstituted in PBS on day of use. Recombinant N, S1 and His tagged RBD proteins were purchased from Genescript (USA). N peptide pool was purchased from Miltenyi Biotec (UK) and RBD peptide pool was purchased from JPT Peptide Technologies (Germany).
TABLE-US-00011 TABLE 2 Covid19 T cell epitopes Predicted binding score coor- (SYFPEITHI) Predicted binding score (IEDB)* Antigen dinates sequence HLA-A2 HLA-A2 HLA-DR1 HLA-DP4 Spike 417-425 KIADYNYKL (SEQ ID NO: 52) 26 0.7 — — glycoprotein 424-433 KLPDDFTGCV (SEQ ID NO: 53) 16 7.4 — — RBD 516-524 ELLHAPATV (SEQ ID NO: 54) 23 3.7 — — 336-355 CPFGEVFNATRFASVTAWNR 17 5.4 28.0 3.17 (SEQ ID NO: 55) 357-376 RISNCVADYSVLYNSASFST 15 3.5 12.0 38.5 (SEQ ID NO: 56) 451-468 YLYRLFRKSNLKPFERDI 20 4.1 22.0 13.2 (SEQ ID NO: 57).sup.¥ 505-524 YQPYRVVVLSFELLHAPATV 23 0.55 0.54 1.0 (SEQ ID NO: 58).sup.¥ Nucleocapsid 138-147 ALNTPKDHI (SEQ ID NO: 59) 22 16.0 — — protein 159-168 LQLPQGTTL (SEQ ID NO: 60) 18 9.0 — — 222-230 LLLDRLNQL (SEQ ID NO: 61).sup.¥ 30 0.8 — — 316-324 GMSRIGMEV (SEQ ID NO: 62) 21 1.8 — — 351-357 ILLNKHIDA (SEQ ID NO: 63).sup.¥ 19 4.5 — — 212-231 GNGGDAALALLLLDRLNQLE 30 0.8 47.0 20.0 (SEQ ID NO: 64) 299-318 KHWPQIAQFAPSASAFFGMS 18 1.5 15.0 30.0 (SEQ ID NO: 65).sup.¥ *The MHCl binding predictions were made on Apr. 03, 2020 using the IEDB analysis resource NetMHCpan (ver. 4.0) tool [184]. .sup.¥Peptides published as known Covid epitopes
TABLE-US-00012 TABLE 3 Cancer antigens T cell epitopes coor- Predicted/ Antigen dinates sequence known binding alleles TRP-2 180-188 SVYDFFVWL (SEQ ID NO: 30) A2 178-192 NCSVYDFFVWLHYYS (SEQ ID NO: 119) A2/A31/A33/A3/DP4 Gp100 44-59 WNRQLYPEWTEAQRLD (SEQ ID NO: 31) DR4 174-190 TGRAMLGTHTMEVTVYH (SEQ ID A2/DR7/DQ6/DR53 NO: 120) 178-186 MLGTHTMEV (SEQ ID NO: 66) A2 476-490 VLYRYGSFSVTLDIV (SEQ ID NO: 67) A1/B35/DP4 NY-ESO-1 157-165 SLLMWITQC (SEQ ID NO: 36) A2 158-166 LLMWITQCF (SEQ ID NO: 35) A24 87-111 LLEFYLAMPFATPMEAELARRSLAQ A24/B35/B52/Cw3/C12/B51/ (SEQ ID NO: 46) DR1/DR4/DP4/DR7/DR9 119-143 PGVLLKEFTVSGNILTIRLTAADHR A68/B49/DR1/DR4/DR52b/DR7/DR8 (SEQ ID NO: 38)
Plasmids
[0271] The backbone of all of the COVID-19 plasmids pVAXDCSN1-SN14 (SN1-15) are derived from the FDA regulatory compliant vector backbone of pVAX1 (Invitrogen) for use in humans. All nucleotide sections for insertion were codon optimised for expression in humans. SN1-SN4 contain a murine IgK leader while SN5-15 contain a human IgH leader. Codon optimised nucleotide sections encoding a leader, amino acids of the S glycoprotein RBD domain 319-541 or 330-525 (Accession Number YP_009724390) alone, fused in frame with either the Hinge-CH2-CH3 domain of the HuIgG1 constant domain (Accession Number P01857) or the variant Hinge-CH2-CH3iV1 (where 23 amino acids have been replaced with murine IgG3 residues) or attached to a fibritin trimer fold on or disulphide bridge motif via a glycine serine linker was synthesised with BamHI and XhoI sites inserted at the 5′ and 3′ends respectively. In first round of cloning these sections were inserted into the BamHI/XhoI sites of the pVAXDCIB68 construct depicted in
[0272] In a second round of cloning codon optimised nucleotide sections encoding a leader, full length nucleoprotein amino acids 2-419 (Accession number YP_009724397) alone or fused in frame with the Hinge-CH2-CH3 domain of the HuIgG1 constant domain or the variant Hinge-CH2-CH3iV1 were synthesised and flanked with HindIII/PstI. The heavy chain was excised using HindIII/PstI from the intermediate vectors generated from the first round and replaced with the N sections in the second expression cassette alongside the appropriate S section depicted in
[0273] To enhance the ImmunoBody vectors pVAXDCIB68 (SCIB1), pVAXDCIB238 (SCIB1plus) and pVAXDCIB178 (SCIB2) the huIgG1 constant region of the antibody heavy chain encoding the CH1-Hinge-CH2-CH3 domains (Acc No: P01857 Amino acid 1-330) was replaced with the same section encoding the replaced 23 murine IgG3 residues at the specific sites. This was achieved by synthesis of the nucleotide section encoding CH1-Hinge-CH2-CH3 iV1 flanked by AfeI and EcoRI. The huigG1 constant domain was excised from the vectors and the section inserted in frame with the heavy variable using these restriction sites.
[0274] The sequences of both chains within each expression cassette of the pVAXDC vector of SN1-15, pVAXDCIB68 iV1, pVAXDCIB238 iV1 and pVAXDCIB178 iV1 was confirmed by the dideoxy chain termination method [185]. DNA nucleotide and translated protein sequences for both chains encoded within plasmids SN1-SN15 and the enhanced ImmunoBody vectors pVAXDCIB68 iV1, pVAXDCIB238 iV1 and pVAXDCIB178 iV1 are shown in
[0275] The plasmid pCMV3-2019-nCoV-Spike (S1+S2)-long utilised encoding full length Spike from SARS-COV2 amino acid 1-1273 (Accession number YP_009724390/QHD43416.1) Was obtained From Sino Biological (catalogue Number VG40589-UT). This contained codon optimised cDNA for expression of the protein in mammalian cells inserted into the KpnI/XbaI sites of the mammalian expression vector pCMV3-untagged under control of the high level expression mammalian human enhanced cytomegalovirus immediate early (CMV) promoter.
[0276] To construct pVAXDCSN16-17 (SN16-17), two consecutive rounds of cloning were required. Two codon optimised nucleotide sections encoding the spike chain comprising of a human IgH leader (MDWIWRILFLVGAATGAHS) and the Spike glycoprotein RBD domain 319-541 amino acids (Accession Number YP_009724390) containing the N501Y mutation from the Kent variant/lineage B.1.1.7, UK-VOC 202012/01 for SN16 or the K417N, E484K and N501Y mutations from the South African variant/lineage VOC 501Y.V2/B1.351 for SN17 flanked by BamHI and XhoI sites at the 5′ and 3′ends respectively were synthesised. In the first round of cloning the sections were inserted into the BamHI/XhoI sites of the pVAXDCIB68 (SCIB1) plasmid in direct replacement of the SCIB1 light kappa chain in the first expression cassette to generate two intermediate plasmids.
[0277] In a second round of cloning two codon optimised nucleotide sections encoding the Nucleoprotein chain comprising of the human IgH leader, full length Nucleoprotein amino acids 2-419 (Accession number YP_009724397) containing either the D3L and S235F mutations from the Kent variant/lineage B.1.1.7, UK-VOC 202012/01 for SN16 or the T2051 mutation from the South African variant/lineage VOC 501Y.V2/B1.351 for SN17, fused in frame with the improved variant Hinge-CH2-CH3 iV1 human IgG1 constant domain (where 23 Amino acids have been replaced with murine IgG3 residues) were synthesised and flanked with HindIII/PstI. The SCIB1 heavy huIgG1 chain was excised using HindIII/PstI from the intermediate plasmids generated from the first round and replaced with the Nucleoprotein section in the second expression cassette alongside the appropriate spike section resulting in SN16 and SN17.
[0278] The sequences of both chains within each expression cassette of the pVAXDC vector for SN16 and SN17 was confirmed by the dideoxy chain termination method. DNA nucleotide and translated protein sequences for both chains encoded within SN16 and SN17 are shown in
TABLE-US-00013 TABLE 4 COVID19 constructs FEATURE AMINO ACID SEQUENCE Murine IgK Leader METDTLLLWVLLLWVPGSTG (SEQ ID NO: 68) Human IgH Leader MDWIWRILFLVGAATGAHS (SEQ ID NO: 69) Spike Glycoprotein RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSA Acc No: YP_009724390 SFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLP (AA 319-541) DDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC NGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLV KNKCVNF (SEQ ID NO: 8) Spike Glycoprotein PNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVS Acc No: YP_009724390 PTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWN (AA 330-525) SNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFP LQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVC (SEQ ID NO: 70) Spike Glycoprotein MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDL Acc No: YP_009724390 FLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGT (AA 1-1273) TLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSS ANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDL PQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYL QPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTE SIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFK CYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGC VIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGF NCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN FNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGG VSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAG CLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAE NSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQY GSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPS KPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPL LTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYE NQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGA ISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAA TKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPA ICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVN NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNE VAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCS CLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT (SEQ ID NO: 71) Fibritin Trimer Fold on GYIPEAPRDGQAYVRKDGEWVLLSTFL (GTGGGSG) (SEQ ID NO: 72) motif Disulphide bridge motif CCGGGSG (SEQ ID NO: 73) Glycine serine Linker GS OR (GGGS) 3GS (SEQ ID NO: 74) OR (GGGS) 3 (SEQ ID NO: 75) HuIgG1 Hinge-CH2-CH3 EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE Acc No: P01857 DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV (AA 99-330) SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK (SEQ ID NO: 76) HuIgG1 Hinge (AA 99-110) EPKSCDKTHTCP (SEQ ID NO: 77) HuIgG1 CH2 (AA 111-223) PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAK (SEQ ID NO: 78) HuIgG1 CH3 (AA 224-330) GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 79) HuIgG1 Hinge-CH2-CH3 EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE iV1 (murine IgG3 23 AA DPEVKFNWYVDGVEVHTAWTQPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV substitutions in bold) SNKALPAPIEKTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAI SVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK (SEQ ID NO: 1) HuIgG1 CH1-Hinge-CH2- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP CH3 AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT iV1 (murine IgG3 23 AA CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV substitutions in bold) DGVEVHTAWTQPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ ID NO: 80) Nucleoprotein SDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTA Acc No: YP_009724397 LTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWY (AA 2-419) FYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQ GTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDA ALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVT QAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVT PSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADET QALPORQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA (SEQ ID NO: 4)
Animals and Cell Lines
[0279] C57Bl/6J, Balb/c (Charles River), HLA-DR4 mice (Model #4149, Taconic), HHDII/HLA-DP4 mice (EM:02221, European Mouse Mutant Archive), HHDII mice (Pasteur Institute) or HHDII/HLA-DR1 mice (Pasteur Institute) aged between 8 and 16 weeks old, were used. All work was carried out with ethical approval under a Home Office approved project license. For all the studies mice were randomised into different groups and not blinded to the investigators.
[0280] Cells, including B16 melanoma expressing relevant MHCI and II alleles (described previously [140, 183, 186, 187]), were cultured in RPMI medium 1640 with L-glutamine (2 mmol/l) with 10% FCS and appropriate antibiotics to maintain plasmids. HEK293T human embryonic kidney cells (ATCC CRL1573) were propagated as described previously [188]. Murine splenocytes were cultured in RPMI-1640 with 10% FBS (Sigma), 2 mM glutamine, 20 mM HEPES buffer, 100 units/ml penicillin, 100 mg/ml streptomycin and 10.sup.−5 M 2-mercaptoethanol. Cell lines utilised were mycoplasma free, authenticated by suppliers (STR profiling), and used within ten passages.
Transient HEK293 Transfection
[0281] Secretion levels from pDNA constructs were evaluated following transient transfections of Expi293F™ cells using the ExpiFectamine™ 293 Transfection kit (Gibco, LifeTechnologies). Briefly, HEK293 cells in suspension (100 ml, 2×10.sup.6/ml) were transfected with 100 μg DNA and conditioned medium harvested at day six post-transfection. Conditioned supernatant was filtered through 0.22 μm bottle top filters (Merck Millipore) and sodium azide added to a final concentration of 0.2% (w/v). Cell pellets were lysed in an appropriate volume of RIPA buffer (Sigma Aldrich, R0278) according to the manufacturer's instruction and clarified by centrifugation prior to analysis.
Immunisation Protocol
[0282] Mice were immunised with 1 μg of DNA via gene gun intradermally on days 1, 8 and 15 or days 1, 15 and 29 and responses analysed on day 21 or 35 respectively unless stated otherwise. For tumour therapy studies mice were implanted with 2.5×10.sup.4 B16F1 or B16 HHDII Nyeso1 tumour cells subcutaneously on day 1 followed by vaccinations on days 4, 11 and 18 or days 4, 8 and 11. Tumours were measured at 3-4 days intervals. Tumour growth in mice was analysed by measuring the tumour size with callipers (length and width). Volume was estimated by the formula below:
Volume=(π/6)×(width×length.sup.2)
Pseudovirus Neutralisation Assays
[0283] SARS-CoV-2 spike protein plasmids were generated and cloned, and pseudoparticles generated following the methodology described for hepatitis C virus in [189]. Pseudoparticles generated in the absence of the plasmid were used as a negative control. For infectivity and neutralisation testing of SARS-CoV-2 pseudoparticles, HEK293T cells per well were plated in white 96-well tissue culture plates (Corning) and incubated overnight at 37° C. The following day, SARS-CoV-2 pseudoparticles were mixed with appropriate amounts of antibody and then incubated for 1 hr at 37° C. before adding to cells. After 72 hrs at 37° C., either 100 μl Bright-Glo (Promega) was added to each well and incubated for 2 mins or cells were lysed with cell lysis buffer (catalog no. E1500; Promega) and placed on a rocker for 15 mins. Luciferase activity was then measured in relative light units (RLUs) using either a SpectraMax M3 microplate reader (Molecular Devices) with the SoftMax Pro6 software (Bright-Glo protocol), or wells were individually injected with 50 μl luciferase substrate and read using a FLUOstar Omega plate reader (BMG Labtech) with the MARS software. Infection by SARS-CoV-2 pseudoparticles was measured in the presence of anti-SARS-CoV-2 mAbs, tested animal sera, preimmune animal sera, and nonspecific IgG at the same dilution. Each sample was tested in duplicate or triplicate. Neutralising activities were reported as 50% inhibitory dilution (ID.sub.50) values and were calculated by nonlinear regression (GraphPad Prism version 7), using lower and upper bounds (0% and 100% inhibition) as constraints to assist curve fitting.
Live Virus Neutralisation Assay
[0284] SARS-CoV-2 infectious virus (CVR-GLA-1) was obtained from the National Centre For AIDS Reagents, NIBSC, UK.
[0285] Live virus neutralisation assays were performed using method previously described [190], except that 790 TCID.sub.50/ml of the SARS-CoV-2 virus was added to each serum dilution. Additionally, for some experiments, the sera were diluted down to 1:81,920.
ACE2 Binding Inhibition Assay
[0286] A V-PLEX COVID-19 ACE2 neutralisation kit from Meso Scale Diagnostics LLC was used to investigate the ability of vaccine-elicited antibodies to block the binding of ACE2 to RBD or whole S proteins. V-plex SARS-CoV-2 Panel 7 multispot plates containing 51 RBD and whole S proteins from Lineage A (originally identified in Wuhan) and variant (B1.1.7, B1.351, P.1) SARS-CoV-2 strains were blocked, followed by incubation with sera at 1:100 dilution and Sulfo-tagged human ACE2 protein, according to the manufacturer's instructions. Results are expressed as percentage inhibition of ACE2 binding via comparison of sera-incubated samples to diluent-containing wells (absence of inhibition).
RBD Binding Inhibition Assay (Surrogate Neutralisation Assay)
[0287] RBD binding inhibition was assessed using kit purchased from Genescript (USA). In brief, sera from immunised mice at various dilutions was mixed with recombinant HRP tagged RBD protein. Mixtures were added to plates precoated with ACE2 receptor and RBD binding detected using TMB substrate. RBD binding inhibition was calculated as loss of colorimetric signal where the negative control was 0%.
ELISpot Assays
[0288] ELISpot assays were performed using murine IFNγ capture and detection reagents according to the manufacturer's instructions (Mabtech AB, Nacka Strand, Sweden). In brief, anti-IFNγ antibodies were coated onto wells of 96-well Immobilin-P plate and quadruplicate wells were seeded with 5×10.sup.5 splenocytes and final concentrations of 10 μg/ml synthetic peptides, 1 μg/ml recombinant protein or 1 μg/ml peptide pools were added unless stated otherwise. Plates were incubated at 37° C. for 40 hrs in an atmosphere of 5% CO.sub.2. Following incubation, captured IFNγ was detected by a biotinylated anti-IFNγ antibody and development with a streptavidin alkaline phosphatase and chromogenic substrate. Spots were analysed and counted using an automated plate reader (Cellular Technologies Ltd Europe GmbH, Aalen, Germany). Functional avidity was calculated as the concentration mediating 50% maximal effector function using a graph of effector function versus peptide concentration.
ELISA for Anti S and N Antibodies
[0289] Commercial N, S1 and RBD proteins (Genescript, USA) were diluted in PBS and coated onto high protein binding 96-well plates at 0.5 μg/well overnight at 4° C. Plates were washed and blocked with casein blocker (Thermo Scientific Ref: 37528) at 200 μl/well for at least an hour at RT, followed by the addition of murine sera (at various dilutions) diluted in PBS 2% BSA for 1 hr at room temperature. Plates were washed and incubated with anti-mouse Ig HRP antibody in PBS 2% BSA (2-step ELISA) or anti-mouse Fc-biotin followed by strepativin-HRPO (3-step ELISA) for 1 hr at room temperature. Following washing TMB substrate added and reaction stopped with 1N H.sub.2SO.sub.4. Commercially available murine IgG N and S1 specific antibodies (Sino Biological) were used as controls (N+ve and S1+ve). Sera from naïve mice was included as a negative control. Absorbance was read at 450 nm wavelength.
Sandwich ELISA for Detection of Secreted RBD and N Proteins
[0290] Commercial kits/antibody pairs were used in both cases. NP was detected in conditioned medium and cell lysate (six days post-transfection) using the SARS-CoV-2 NP ELISA kit from Bioss, (cat #BSKV0001) according to supplier's instructions. Quantitation relied on the standard curve with NP standard supplied by the kit. RBD (secreted and in cell lysate) was detected using a sandwich ELISA consisting of a capture antibody: SARS-CoV-2 Spike neutralising mouse mAb (Sino Biological, 40591-MM43) combined with a HRPO-labelled detection antibody from the SARS-CoV-2 S protein RBD Antibody Pair (Epigentek A73682). Capture antibody was coated at 200 ng/well; detection antibody was used at a dilution of 1:1000.
SPR Analysis of CD64 Binding by Fc Fusion Constructs
[0291] The analysis was performed on a BiaT200. His-tagged CD64 (Acrobiosystems, FCA-H52H1) was captured onto a CM5 chip coupled with an anti-His antibody. The chip contains 4 flow cells, three of which were used to capture increased densities of CD64, the fourth flow cell was a reference cell. Fc-constructs were titrated over 50.0 nM to 0.78 nM concentration range, and the association and dissociation monitored for 210 s and 700 s respectively, at a flow rate of 30 ul/′. Kinetic parameters were deduced by fitting the data to a 1:1 monovalent binding model.
Immunogenicity Analysis of the Modified Fc (iFcv1) Construct
[0292] The analysis was performed by Abzena (Cambridge) Ltd according to HTA standards. PBMCs were isolated from healthy community donor buffy coats (obtained under consent from commercial vendors). Cells were separated by density centrifugation using Lymphocyte separation medium (Corning, Amsterdam, The Netherlands) and CD8+ T cells were depleted using CD8+ RosetteSep™ (StemCell Technologies Inc, London, UK). Donors were characterised by identifying HLA-DR and HLA-DQ haplotypes to 4digit resolution by HISTO Spot SSO HLA typing (MC Diagnostics, St. Asaph, UK). T cell responses to the neo-antigen KLH (Invitrogen, Paisley, UK) were also determined. PBMC were then frozen and stored in the vapour phase of nitrogen until required. A cohort of up to 50 donors was selected covering 77% of HLA alleles. PBMCs from each donor were thawed, counted and viability assessed using trypan blue (Merck Life Science UK Ltd, Gillingham, UK) dye exclusion. For each donor, bulk cultures were established in which 1 ml of the proliferation cell stock was added to the appropriate wells of a 24 well plate. 1 ml of the test sample (iTv1) was added to the PBMC to give a final sample concentration of 0.3 μM. For each donor, a reproducibility control well (cells incubated with 0.3 μM KLH), a clinical benchmark control well (cells incubated with 5 μM Bydureon®), a low immunogenicity control (cells incubated with 0.3 μM Herceptin®) and a culture medium only well were also included. Cultures were incubated for a total of 8 days at 37° C. with 5% CO.sub.2. On days 5, 6, 7 and 8, the cells in each well were gently resuspended by mixing 5× using an electronic pipette and 3×100 μl aliquots transferred to each well of a round bottomed 96 well plate. The cultures were pulsed with 0.75 μCi [3H]-Thymidine (Perkin Elmer®, Beaconsfield, UK) in 100 μl AIM-V® culture medium and incubated for a further 18 hours before harvesting onto filter mats (Perkin Elmer®, Beaconsfield, UK) using a TomTec Mach III cell harvester. CPM for each well were determined by Meltilex™ (Perkin Elmer®, Beaconsfield, UK) scintillation counting on a 1450 Microbeta Wallac Trilux Liquid Scintillation Counter (Perkin Elmer®, Beaconsfield, UK) in paralux, low background counting. An empirical threshold of a SI equal to or greater than 1.9 (SI ≥90) has been previously established whereby samples inducing responses above this threshold were deemed positive.
Statistical Analysis
[0293] Statistical analysis of responses to the nucleic acid plasmids was performed using Graph Pad Prism software version 7. Comparative analysis of the ELISpot results was performed by applying paired or unpaired ANOVA or Student t test as appropriate with p values calculated accordingly. Comparison of avidity curves/survival was assessed by applying the F test using the GraphPad Prism software, or log-rank test. P<0.05 values were considered statistically significant.
[0294] Statistical analysis and comparison of neutralisation titres between groups (ID.sub.50 values) was performed using Kruskal-Wallis analysis of variance (ANOVA) with Dunn's multiple-comparison test. Data analysis was not blinded. As above, differences were considered statistically significant at a p value of <0.05 and statistical analyses performed using the GraphPad Prism 8 software.
Preclinical Studies Example 1—RBD Protein and N Protein Secretion from Transient HEK293 Transfections with pDNA
[0295] In order to assess the transfection efficiency of the pDNA constructs, HEK293 cells were transiently transfected with the pDNA using Thermofisher's Expi293 system and protein secretion in the medium evaluated using sandwich ELISAs for the RBD protein and Nucleoprotein (
Example 2—T Cell Responses to the RBD and N Proteins (NP) with pDNA Delivered Via Gene Gun to HHDII Mice
[0296] T cell responses to pVAXDC SPIKE RBD+NP (SN8), pVAXDC SPIKE RBD v2 TRIMER+NPFC (SN9), pVAXDC SPIKE RBD v3 TRIMER+NPFC (SN10) and pVAXDC SPIKE RBD v2 TRIMER+NPFC iV1 (SN11) following three weekly immunisations of HHDII mice with pDNA administered via gene gun were measured. The frequency of the IFNγ ELISpot responses to all RBD constructs was measured using predicted or previously identified T cell epitopes and the whole S1 protein, the RBD recombinant protein and RBD peptide pool. All four constructs showed strong responses to the S1 protein as well as the RBD peptide pool and RBD aa417-425 peptide of which responses to RBD peptide pool and RBD aa417-425 peptide reached significance from all constructs (
[0297] Another study compared responses to pVAXDC SPIKE RBD+NP (SN8), pVAXDC SPIKE RBD v3 TRIMER+NPFC (SN10) and pVAXDC SPIKE RBD v2 TRIMER+NPFC iV1 (SN11) following three weekly immunisations of HHDII mice with pDNA administered via gene gun. All three constructs showed significant responses to the S1 protein as well as the RBD peptide pool but only SN11 immunised mice showed significant responses to RBD aa417-425 peptide (
Example 3—T Cell Responses to the RBD and N Proteins (NP) with pDNA Delivered Via Gene Gun to HHDII/DR1 Mice
[0298] T cell responses to pVAXDC SPIKE RBD+NP (SN8), pVAXDC SPIKE RBD v2 TRIMER+NPFC (SN9), pVAXDC SPIKE RBD v3 TRIMER+NPFC (SN10) and pVAXDC SPIKE RBD v2 TRIMER+NPFC iV1 (SN11) following three weekly immunisations of HHDII/DR1 mice with pDNA administered via gene gun were measured. The frequency of the IFNγ ELISpot responses to all RBD constructs was measured using an identified T cell epitope (RBD aa417-425), the whole S1 protein, whole RBD protein and the RBD peptide pool. Constructs SN8, SN9 and SN10 showed significant responses only to the whole S1 protein whereas construct SN11 generated responses to the S1 and RBD proteins, RBD peptide pool and RBD aa417-425 peptide. (
Example 4—Construct Containing NPFC iV1 Generates Higher Frequency Responses to N Protein
[0299] To compare the T cell responses from constructs containing N antigen (SN8), N linked to FC (SN9) or N linked to modified iV1 FC (SN11) data was combined and normalised against background control. Construct SN11 showed significantly enhanced responses to both N protein and the N 138-147 peptide when compared to SN8 or SN9 (** p<0.01 and * p<0.05 respectively) (
Example 5—T Cell Responses to the RBD and N Proteins with pDNA Delivered Via Gene Gun to HHDII/DP4 Mice
[0300] T cell responses to pVAXDC SPIKE RBD TRIMER+NPFC (SN2), pVAXDC SPIKE RBD FC+NP (SN3) and pVAXDC SPIKE RBD+NP (SN4) following three fortnightly immunisations of HHDII/DP4 mice with pDNA administered via gene gun were measured. The frequency of the IFNγ ELISpot responses to all RBD constructs was measured using predicted and identified T cell epitopes, the whole S1 protein and the RBD protein. Significant responses were seen from construct SN2 to the RBD aa417-425 peptide, RBD protein and RBD peptide pool and from construct SN4 to the S1 and RBD proteins (
Example 6—Avidity of T Cell Responses to the S1 Protein with pDNA Delivered Via Gene Gun is Superior from RBD FC and RBD TRIMER Constructs
[0301] T cell responses to pVAXDC SPIKE RBD TRIMER+NPFC (SN2), pVAXDC SPIKE RBD FC+NP (SN3) and pVAXDC SPIKE RBD+NP (SN4) following three fortnightly immunisations of HHDII/DP4 mice with pDNA administered via gene gun were assessed for avidity to S1 protein titration. Responses in mice immunised with SN2 and SN3 show significantly higher avidity of responses (p<0.0001) compared to those immunised with SN4 (
Example 7—pDNA Immunisation Generates High Avidity Peptide Specific Responses to the RBD 417-425 Epitope
[0302] T cell responses to pVAXDC SPIKE RBD v2 TRIMER+NPFC iV1 (SN11) following three weekly immunisations of HHDII mice with pDNAs administered via gene gun were assessed for avidity to RBD 417-425 peptide by peptide titration. Responses in mice immunised with SN11 show high avidity responses of greater than 0.0001 ug/ml (
Example 8—Frequency and Avidity of T Cell Responses to the N 138-146 Peptide with pDNA Delivered Via Gene Gun is Superior from NPFC iV1 Construct
[0303] T cell responses to pVAXDC SPIKE RBD v3 TRIMER+NPFC (SN10), pVAXDC SPIKE RBD v2 TRIMER+NPFC iV1 (SN11) following three weekly immunisations of HHDII mice with pDNA administered via gene gun were assessed for avidity to N 138-146 peptide by peptide titration. Responses in mice immunised with SN11 show higher frequency as well as slightly higher avidity of responses compared to those immunised with SN10 (
Example 9—Antibody Responses to the RBD and N Proteins with pDNA Delivered Via Gene Gun
[0304] Antibody responses to pVAXDC SPIKE RBD+NPFC (SN5), pVAXDC SPIKE RBD TRIMER+NPFC (SN6), pVAXDC SPIKE RBD v2 TRIMER+NPFC (SN9), pVAXDC SPIKE RBD v3 TRIMER+NPFC (SN10), pVAXDC SPIKE RBD v2 TRIMER+NPFC iV1 (SN11) or pVAXDC SPIKE RBD TRIMER+NPFC (SN2), pVAXDC SPIKE RBD FC+NP (SN3) and pVAXDC SPIKE RBD+NP (SN4) following three fortnightly immunisations of HHDII/DP4 mice with pDNA administered via gene gun were measured. The Ab titres to the S1, RBD and N proteins were compared in sera from mice immunised with the monomer RBD construct, the dimer RBD presented as an Fc fusion protein, the RBD construct as a trimer and the shorter RBD as a trimer both presented as a fibritin construct. Antibodies were assessed in sera at 1/100 to 1/10,000 dilution. Strong reactivity to the N protein was observed in sera from all immunised mice even at 1/10,000 dilution (
[0305] Antibody responses to pVAXDC SPIKE RBD FC+NP (SN3), pVAXDC SPIKE RBD+NP (SN8), pVAXDC SPIKE RBD v3 TRIMER+NPFC (SN10) and pVAXDC SPIKE RBD v2 TRIMER+NPFC iV1 (SN11) following three weekly immunisations of HHDII mice with pDNA administered via gene gun were measured in an ELISA assay. Antibodies were assessed in sera at 1/100 to 1/10,000 dilution. Strong reactivity to the N protein was observed in sera from all immunised mice even at 1/10,000 dilution. Reactivity to the S1 protein was seen at 1/100 and 1/1000 dilutions of sera and was highest from construct SN10 containing the shorter RBD v3 trimer (SN10) but also detectable at 1/1000 sera dilution from construct SN8 containing the full length RBD monomer (
Example 10—pDNA Delivered Via Gene Gun Elicits Virus Neutralising Antibody Responses with Similar Titre to Total Antibody Measurement
[0306] Virus neutralising antibodies were assessed in a surrogate neutralisation assay for inhibition of RBD binding to plate bound ACE2 receptor. Sera from mice immunised with constructs SN5, SN6 and SN10 containing the longer RBD monomer, longer RBD trimer or shorter RBD v3 trimer, respectively, showed >50% inhibition at 1/100 dilution with lower titres seen from constructs SN9 and SN11 (
[0307] Virus neutralisation was also analysed at different sera dilutions in the pseudovirus neutralisation assay (
TABLE-US-00014 TABLE 5 Summary of COVID19 constructs Name Description (1-4 murine Leader IgK) SN1 pVAXDC Spike RBD + NPFC SN2 pVAXDC SPIKE RBD TRIMER + NPFC SN3 pVAXDC SPIKE RBD FC + NP SN4 pVAXDC SPIKE RBD + NP (5-15 Human Leader IgH) SN5 pVAXDC HuL SPIKE RBD + HuL NPFC SN6 pVAXDC HuL SPIKE RBD TRIMER + HuL NPFC SN7 pVAXDC HuL SPIKE RBD FC + HuL NP SN8 pVAXDC HuL SPIKE RBD + HuL NP SN9 pVAXDC MOD HuL Spike RBD v2 TRIMER + HuL NPFC SN10 pVAXDC MOD HuL Spike RBD v3 TRIMER + HuL NPFC SN11 pVAXDC MOD HuL Spike RBD v2 TRIMER + HuL NPFC iV1 SN12 pVAXDC HuL SPIKE RBD TRIMER + HuL NPFC iV1 SN13 pVAXDC HuL SPIKE RBD FC + HuL NPFC iV1 SN14 pVAXDC HuL SPIKE RBD FC iV1 + HuL NPFC iV1 SN15 pVAXDC HuL SPIKE RBD + HuL NPFC iV1 SN16 pVAXDC HuL SPIKE RBD (B1.1.7 variant) + HuL NPFC iV1 (B1.1.7 variant) SN17 pVAXDC HuL SPIKE RBD (B1.351 variant) + HuL NPFC iV1 (B1.351 variant)
TABLE-US-00015 TABLE 6 Summary of immune responses to COVID-19 constructs N protein RBD T cell T cell T cells T cells T cells to N to 138 to RBD to S1 to 417 p.Virus (spots/ (spots/ (spots/ (spots/ (spots/ Avidity Abs Abs ACE VNAbs con- Secretion 10{circumflex over ( )}6 10{circumflex over ( )}6 con- secretion 10{circumflex over ( )}6 10{circumflex over ( )}6 10{circumflex over ( )}6 to 417 To RBD to S1 VNAbs % inh SN struct (μg/ml) cells) cells) struct (μg/ml) cells) cells) cells) (ng/ml) (titre) (titre) (titre) at 1/100 1 Fc M 2 Fc 1 T 10 300 300 200 0.1 30 3 M 14 Fc 200 400 600 200 0.0008 100 80 4 M 18 M 150 200 300 170 15 5 Fc 2 M 150 10,000 1000 100 90 6 Fc 1 T 10 1,000 100 100 60 7 M 5 Fc 600 8 M 5 300 50 m 100 400 100 400 1000 9 Fc 0.5 310 50 St 10 500 100 400 100 100 10 40 10 Fc 1 450 100 Cc 20 300 1000 100/1000 100 45 11 Fcv1 0.5 700 450 st 10 750 700 1,000 100 100 10 30 12 Fcv1 0.37 210 550 T 54 740 200 780 10000 60 13 Fcv1 0.16 170 385 Fc 408 465 85 500 10000 50 14 Fcv1 0.16 270 720 Fcv1 152 780 220 895 10000 90 15 Fcv1 0.32 160 700 M 195 880 200 900 10000 90 16 Fcv1 142 M 588 152 30000 17 Fcv1 563 M 673 367 10-100000 30-100000 25-98 M monomer, Fc Fc-fusion, Fcv1 Fc-fusion enhanced, T trimer, sm small monomer, st small trimer cc-extra cysteines
[0308] All COVID-19 constructs contained a S protein RBD, either as a monomer a trimer or an Fc fusion protein and an N protein, either as a monomer, an Fc fusion protein or an Fc fusion protein modified to allow non covalent association of antigen-Fc fusion protein at the cell surface (Tables 5 and 6).
[0309] SN11, which expresses the N protein fused to modified Fc gave significantly better T-cell responses to N protein and to the HLA-A2 epitope N 138-146 than N protein fused to unmodified Fc or to the N protein alone. Of even more interest was that this construct also gave superior responses to RBD, S1 and peptide RBD 417-425 than a similar construct expressing the same RBD construct but N-Fc. This suggests that the modified N-Fc is acting like an adjuvant and activating the APCs to also enhance the T-cell response to other antigens.
[0310] In contrast, the best VNAbs were stimulated to the RBD monomer (SN1, SN4, SN5, SN8, SN15), the RBD trimer (SN2, SN6, SN9, SN11, SN12) and the RBD-Fc (SN3, SN7, SN13, SN14). Constructs were produced comparing the RBD trimer to the RBD-Fc and the RBD-enhanced Fc in combination with the Fc modified N protein. The constructs containing either the RBD monomer and N protein fused separately to enhanced Fc regions (SN15) or the RBD and N protein fused separately to enhanced Fc regions (SN14) produced the strongest antibody and T cell responses.
[0311] The examples above show a vaccine that incorporates the RBD of the spike protein to stimulate neutralising antibodies and T cell responses but also the N protein to induce memory T cell responses that will confer protection against not only COVID19 but also any new emerging coronaviruses as the N protein is highly conserved and rarely mutates.
Example 11. pDNA Encoding T Cell Epitopes within CDRs of Fc Modified HuIgG1 Construct (iSCIB1) Generate Strong T Cell Responses
[0312] Conventional C57Bl/6 or HLA transgenic mice (HLA-DR4) were immunised with pDNA encoding SCIB1 (WO2008/116937—
Example 12. pDNA Encoding T Cell Epitopes within CDRs of Fc Modified HuIgG1 Construct (iSCIB1plus) Generate Strong T Cell Responses
[0313] HLA transgenic mice (HLA-DR4, C57Bl/6 or HHDII/DP4) were immunised with pDNA encoding SCIB1plus (
Example 13. pDNA Encoding T Cell Epitopes within CDRs of Fc Modified HuIgG1 Constructs (iSCIB1 and iSCIB1plus) Mediate Efficient Tumour Therapy
[0314] Conventional C57Bl/6 or HLA transgenic mice (HHDII/DP4) were implanted with B16 melanoma cells expressing the appropriate MHC alleles followed by immunisation with pDNA encoding SCIB1 (
Example 14. pDNA Encoding T Cell Epitopes within CDRs of Fc Modified HuIgG1 Construct (iSCIB2) Generate Strong T Cell Responses
[0315] HLA transgenic mice (HHDII or HHDII/DR1) were immunised with pDNA encoding SCIB2 (
Example 15. pDNA Encoding T Cell Epitopes within CDRs of Fc Modified HuIgG1 Constructs (iSCIB2) Mediate Efficient Tumour Therapy
[0316] HLA transgenic mice (HHDII) were implanted with B16 melanoma cells expressing the appropriate MHCI allele followed by immunisation with pDNA encoding SCIB2 (
Example 16. COVID-19 Specific T Cell and Neutralising Antibody Responses from pDNA Delivered Via Gene Gun is Superior from RBD Fc iV1 Construct Compared to RBD Fc Construct
[0317] Balb/c and HLA-A2 transgenic mice were immunised with pDNA containing N protein fused to modified Fc (NPFC iV1) alongside either the RBD domain fused to Fc (RBD FC, SN13,
[0318] Antibody responses in immunised Balb/c mice were assessed by ELISA and showed similar titres of antibodies specific for the S1 protein in SN13 and SN14 immunised mice (
Example 17. RBD Protein and N Protein Secretion from Transient HEK293 Transfections with SN11, 12, 13, 14 and 15 pDNA
[0319] In order to assess the transfection efficiency of the SN11, 12, 13, 14 and 15 pDNA constructs compared to SN5 pDNA, HEK293 cells were transiently transfected with the pDNA using Thermofisher's Expi293 system and protein secretion in the medium and cell lysates evaluated using sandwich ELISAs for the RBD protein and Nucleoprotein
Example 18. COVID-19 Specific Neutralising Antibody Responses from SN15 pDNA Delivered Via Gene Gun is Superior to that from Whole S pDNA
[0320] Balb/c and C57Bl/6 mice were immunised with pDNA containing N protein fused to modified Fc (NPFC iV1) alongside either the RBD monomer (SN15), RBD trimer (SN12) or RBD monomer linked to Fc (SN13) or a with a whole S pDNA. Antibody responses in immunised Balb/c mice were assessed by ELISA and showed higher titres of antibodies and total IgG specific for the S1 protein in SN15 and SN13 compared to whole S DNA or SN11 immunised mice (
Example 19. COVID-19 Specific T Cell Responses from SN15 pDNA Delivered Via Gene Gun are Superior to that from Whole S pDNA
[0321] Balb/c, HLA-A2 transgenic and C57Bl/6 mice were immunised with pDNA containing N protein fused to modified Fc (NPFC iV1) alongside either the RBD monomer (SN15), RBD trimer (SN12), RBD short trimer (SN11) or RBD monomer linked to Fc (SN13) or a with a whole S pDNA. T cell responses in immunised mice were assessed by IFNγ ELISpot assay and showed high frequency responses to a pool of overlapping peptides from the RBD protein in mice immunised with SN15, SN13, SN12 and SN11 pDNA (
Example 20. Fc Modified HuIgG1 Constructs Show More Avid CD64 Binding Compared to Unmodified Fc
[0322] Targeting antigens to the high affinity FcgammaR1 (CD64) induces better humoral and T cell responses through a combination of enhanced antigen internalisation as well as improved APC activation. A purified RBD construct with a modified Fc shows prolonged interaction with CD64 compared to the unmodified RBD-Fc construct, a phenomenon more pronounced at higher CD64 receptor densities, suggesting increased avidity (
TABLE-US-00016 TABLE 7 kinetic constants of the interaction of Fc-containing constructs (Bia T200) Receptor Density ka kd KD Chi.sup.2 construct (RU) (1/Ms) (1/s) (M) RMAX (RU.sup.2) RBD-Fc ~30 1.98E+05 7.97E−04 4.02E−09 32.9 2.76 ~300 2.26E+05 1.06E−03 4.68E−09 546.3 92.6 ~900 2.31E+05 1.05E−03 4.57E−09 1033.1 347 RBD-iFcv1 ~30 2.60E+05 6.08E−04 2.34E−09 47.6 1.7 ~300 1.89E+05 3.05E−04 1.61E−09 917.3 37.7 ~900 2.00E+05 1.93E−04 9.63E−10 1592.3 180
Example 21. Lack of Immunogenicity of the Modified iFcv1 Construct in a 20-Donor Panel
[0323] The presence of residue changes in the modified Fc (iFcv1) theoretically has the capacity to induce immunogenicity when administered in the clinic to human volunteers. This was therefore assessed in studies conducted by Abzena (UK) using trastuzumab (Herceptin®) as the comparator. To assess whether the iFcv1 fusion construct has the potential to induce CD4 T cell responses in humans, an important driver of immunogenicity, a trastuzumab construct containing the modified iFcv1 was created: ‘iTv1’. The immunogenicity iTv1 was assessed in a proliferation assay (3H-thymidine uptake) using CD8 depleted peripheral blood mononuclear cells (PBMCs) from a panel of 20 donors representing the European and North American population, covering approximately 77% of HLA alleles. The T cell responses were assessed on days 5-8 following incubation with iTv1 or wild-type Trastuzumab (Herceptin®) and Bydureon® controls (
Example 22. Lack of Immunogenicity of the Modified iFcv1 Construct in a 50-Donor Panel
[0324] In addition to the study performed in a 20-donor panel a repeat was performed to extend to a 50 donor panel spanning a broader range of HLA types. The modified Fc construct, iTv1, generated a small increase in proliferation in 3/50 donors; this is again comparable to the results seen with Abzena's low immunogenicity control Herceptin®, where a small increase in proliferation was observed in 2/20 donors and a larger increase in 1/50 donors (
Example 23. COVID-19 N Protein Specific Antibody Responses are Elicited from pDNA Encoding NP, NP Fc and NP FciV1 Delivered Via Gene Gun
[0325] Sera from mice immunised with NP, NP Fc or NP FciV1 encoding pDNA were analysed for antibody reactivity to SARS-CoV-2 N protein by ELISA. Similar antibody responses and EC.sub.50 values were seen from all constructs irrespective of whether it was fused to Fc or not (
Example 24. Antibody Responses Cross Reactive with Variant Strains are Induced by SN15 pDNA Vaccination
[0326] Balb/c mice were immunised with pDNA containing N protein fused to modified Fc (NPFC iV1) alongside the RBD monomer (SN15) and antibody responses in sera were assessed by ELISA to variant S1 proteins from Wuhan (Lineage A), B.1.351 and B.1.1.7 virus strains. Higher titres of antibodies were observed specific for the S1 protein from lineage A, B.1.1.7 and B.1.351 variants with detectable responses over background at down to 1 in 100,000 sera dilution. No significant difference in the reactivity to Lineage A and B.1.1.7 variant S1 proteins was seen (
Example 25. Antibody Responses Cross Reactive with Variant Strains are Induced by SN15, SN16 and SN17 pDNA Vaccination that Also Inhibit ACE2 Binding
[0327] Balb/c mice were immunised with pDNA containing the RBD monomer (SN15), RBD monomer from B.1.1.7 variant (SN16,
[0328] The ability of sera from immunised mice to inhibit the binding of the ACE2 receptor to the variant RBD or whole S proteins was assessed using the MesoScale Discovery platform. Inhibition of RBD binding to ACE2 was higher for sera from mice vaccinated with the original Lineage A vaccine construct (SN15) and the B.1.351 variant vaccine construct (SN17) compared to that seen with the NIBSC 20/136 control (
Example 26. Sera from SN15 and SN17 Immunised Mice Show Virus Neutralisation in Pseudotype and Live Virus Neutralisation Assays
[0329] Sera from Balb/c mice immunised with the original Lineage A (SN15) or 6.1.351(5N17) variant vaccines were also assessed in pseudotype and live virus neutralisation tests against the original Lineage A and B.1.351 variants. Sera from mice immunised with the original variant vaccine showed potent neutralisation of the original Lineage A pseudotype, with reduced efficacy against the B.1.351 variant vaccine (ID.sub.50 values of 6232 and 2137 respectively) (
Example 27. T Cell Responses are not Impacted by Variations in the Virus Strains
[0330] To examine if the T cell responses were influenced by the mutations in the different virus variants, splenocytes from mice immunised with either the original Lineage A vaccine (SN15) or the B.1.351 vaccine (SN17) were stimulated ex vivo with RBD and N peptide pools derived from the original sequence. T cell responses specific for RBD and N were detected with little difference between the response induced by the different vaccine constructs (
Example 28. Antibody Responses can be Efficiently Boosted by Vaccines Specific for Variant Strains and Show Cross Reactivity to B.1.351 and B.1.617.2 Variants
[0331] To examine if antibody responses elicited against the Lineage A virus with the SN15 vaccine could be boosted by a vaccine targeting the B.1.351 variant (SN17) Balb/c mice were immunised with the SN15 vaccine on days 1 and 29 followed by a boost at day 85 with SN17 vaccine. Antibody responses in sera samples taken at days 42, 82 and 98 were examined by ELISA for reactivity to the Lineage A and B.1.351 S1 proteins. As a comparison, mice were immunised with only SN17 vaccine. Antibody responses are detectable in both sets of sera down to 1 in 100,000 dilution (
[0332] Sera from mice immunised with these prime boost regimes were analysed for reactivity to the RBD proteins from the B.1.351 and 6.1.617.2 variants by ELISA (
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