CN117295761A - Antibodies conjugated or fused to receptor binding domains of SARS-COV-2 spike protein and their use for vaccine purposes - Google Patents

Abstract

A SARS‑CoV‑2 vaccine is critical to reducing morbidity and mortality. The inventors generated an antibody against a surface antigen (i.e., CD40) on antigen-presenting cells (i.e., dendritic cells) in which the heavy chain was coupled to the receptor binding domain of the Sars-Cov-2 spike protein for Its use as a vaccine. Specifically, the inventors show that the vaccine induces circulating antibody-secreting hu-B cells, elicits S-specific IgG+ hu-B cells, causes expansion of central memory CD4+ hu-T cells and effector memory CD4 The emergence of +T cells causes the expansion of central memory CD8+hu‑T cells and the emergence of effector memory CD8+T cells, ultimately inducing stem cell-like memory hu‑CD8+T cells. Accordingly, the present invention relates to antibodies directed against surface antigens of antigen-presenting cells, wherein the heavy and/or light chains are coupled or fused to the receptor binding domain of the Sars-Cov-2 spike protein.

Description

Antibodies coupled or fused to the receptor binding domain of the SARS-COV-2 spike protein and their use for vaccine purposes

Technical Field

The present invention relates to the field of medicine, in particular to the fields of virology and vaccinology.

Background Art

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a global health threat. On March 11, 2020, the World Health Organization declared COVID-19 a pandemic. The speed, rate, and observed increased fatality rate of global transmission present public health, socioeconomic, and scientific challenges. As its transmission appears to be very active, as of May 10, 2020, more than 185 countries have been infected, with more than 4,100,000 confirmed cases and more than 280,000 deaths. SARS-CoV-2 can cause a respiratory syndrome with clinical pathology similar to mild upper respiratory tract illness (common cold-like symptoms) and occasionally severe lower respiratory tract disease and extrapulmonary manifestations, leading to multi-organ failure and death. This pandemic follows several highly pathogenic human coronavirus infections, including SARS-CoV in 2002, with a fatality rate of 10%, and MERS-CoV in 2012, with a fatality rate of 36%. No treatment or vaccine is available. However, if the virus becomes established in the human population, a SARS-CoV-2 vaccine will be critical to reducing morbidity and mortality.

Summary of the invention

The invention is defined by the claims. In particular, the invention relates to antibodies against surface antigens of antigen presenting cells, wherein the heavy chain and/or light chain is coupled or fused to the receptor binding domain of the Sars-Cov-2 spike protein.

DETAILED DESCRIPTION

definition:

As used herein, the term "subject" or "a subject in need thereof" refers to a human or non-human mammal. Typically, the patient has or may be infected with SARS-Cov-2.

As used herein, the term "coronavirus" has its ordinary meaning in the art and refers to any member of the Coronaviridae family. Coronaviruses are positive-strand RNA viruses with a genome that is approximately 27 kb to approximately 33 kb long (depending on the specific virus). The virion RNA has a cap at the 5' end and a poly A tail at the 3' end. The length of the RNA makes coronaviruses the largest of the RNA virus genomes. In particular, coronavirus RNA encodes: (1) an RNA-dependent RNA polymerase; (2) an N protein; (3) three envelope glycoproteins; and (4) three nonstructural proteins. These coronaviruses infect a variety of mammals and birds. They cause respiratory infections (common), enteric infections (mainly in infants >12 months of age), and may cause neurological syndromes. Coronaviruses are transmitted via aerosols of respiratory secretions.

As used herein, the term "severe acute respiratory syndrome coronavirus 2" or "SARS-Cov-2" has its ordinary meaning in the art and refers to the strain of coronavirus that causes coronavirus disease 2019 (COVID-19), which refers to a respiratory syndrome with clinical pathological manifestations similar to mild upper respiratory tract illness (common cold-like symptoms), and occasional severe lower respiratory tract disease and extrapulmonary manifestations, leading to multi-organ failure and death. In particular, the term refers to severe acute respiratory syndrome coronavirus 2 isolate 2019-nCoV_HKU-SZ-005b_2020, whose complete genome is available under NCBI accession number MN975262.

As used herein, the term "Covid-19" refers to the respiratory disease caused by severe acute respiratory syndrome coronavirus 2.

As used herein, the term "asymptomatic" refers to a subject who does not experience detectable symptoms of coronavirus infection. As used herein, the term "symptomatic" refers to a subject who experiences detectable symptoms of coronavirus infection. Symptoms of coronavirus infection include: fatigue, loss of smell, headache, cough, fever, and difficulty breathing.

As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to amino acid polymers of any length. These terms also include amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. When discussed in the context of gene therapy, a polypeptide refers to the corresponding intact polypeptide, or any fragment or genetically engineered derivative thereof that retains the desired biochemical function of the intact protein.

As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, the nucleotide structure may be modified before or after polymer assembly. As used herein, the term polynucleotide refers to double-stranded and single-stranded molecules interchangeably. Unless otherwise specified or required, any embodiment of the present invention as described herein is a polynucleotide, which includes a double-stranded form, and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

As used herein, the expression "derived from" refers to a process whereby a first component (e.g., a first polypeptide) or information from the first component is used to isolate, derive or prepare a different second component (e.g., a second polypeptide different from the first polypeptide).

As used herein, "percent identity" between two sequences is a function of the number of identical positions that the sequences have (i.e., the total number of positions × 100 of identity% = number of identical positions/positions), taking into account the number of spaces and the length of each space that need to be introduced to achieve the best alignment of the two sequences. The sequence comparison between the two sequences and the determination of the percent identity can be achieved using a mathematical algorithm as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970) "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443–53.). The percent identity between two nucleotide or amino acid sequences can also be determined using an algorithm such as EMBOSS Needle (pairwise alignment; Available at www.ebi.ac.uk). For example, EMBOSS Needle can be used with the BLOSUM62 matrix, with a "gap open penalty" of 10, a "gap extension penalty" of 0.5, an incorrect "end point gap penalty", an "end point gap open penalty" of 10, and an "end point gap extension penalty" of 0.5. In general, "percent identity" is a function of the number of matching positions divided by the number of comparison positions multiplied by 100. For example, if after alignment, 6 out of 10 sequence positions are identical between the two comparison sequences, the identity is 60%. % identity is usually determined over the entire length of the query sequence being analyzed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical, regardless of any chemical and/or biological modification. According to the present invention, a first amino acid sequence having at least 90% identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the second amino acid sequence.

As used herein, the term "mutation" has its general meaning in the art, referring to substitution, deletion or insertion. In particular, the term "substitution" refers to the removal of a specific amino acid residue at a specific position and the insertion of another amino acid residue at the same position. In the specification, mutation reference is made according to standard mutation nomenclature. In particular, the term "mutation" includes "naturally occurring mutations" and "non-naturally occurring mutations".

As used herein, the term "naturally occurring mutation" refers to any mutation that can be found in naturally occurring variants of SARS-CoV-2 polypeptides, generally including the B.1.1.7 lineage (also known as 20I/501Y.V1 variant of concern (VOC) 202012/01), the B.1.351 lineage (also known as 20H/501Y.V2), and the P.1 lineage (also known as 20J/501Y.V3). Such mutations are well known in the art, including those described in the following references incorporated by reference:

·(1)Jie Hu et al.The D614G mutation of SARS-CoV-2spike proteinenhances viral infectivity and decreases neutralization sensitivity toindividual convalescent sera.bioRxviv(2020)

·(2)Korber B.et al.Spike mutation pipeline reveals the emergence of a more transmissible form of SARS-CoV-2.bioRxviv(2020).doi.org/10.1101/2020.04.29.069054.

·(3)Lizhou Zhang et al.The D614G mutation in the SARS-CoV-2spikeprotein reduces S1 shedding and increases infectivity.bioRxviv(2020).doi.org/10.1101/2020.06.12.148726.

·(4)Junxian Ou et al.Emergence of RBD mutations in circulating SARS-CoV-2strains enhancing the structural stability and human ACE2 receptoraffinity of the spike protein.bioRxiv(2020).doi:10.1101/2020.03.15.991844v4

·(5)Saha, P.et al.Mutations in Spike Protein of SARS-CoV-2ModulateReceptor Binding, Membrane Fusion and Immunogenicity: An Insight into ViralTropism and Pathogenesis of COVID-19.chemRxiv(2020)doi:10.26434/chemrxiv.12320567 .vl

·(6)Jian Shang, Yushun Wan, Chuming Luo, Gang Ye, Qibin Geng, AshleyAuerbach, Fang Li. Cell entry mechanisms of SARS-CoV-2Proceedings of the National Academy of Sciences May 2020, 117(21)11727-11734; DOI ：10.1073/pnas.2003138117

·(7)Allison J. Greaney, Andrea N. Loes, Katharine H. D. Crawford, Tyler N. Starr, Keara D. Malone, Helen Y. Chu, Jesse D. Bloom, bioRxiv 2020.12.31.425021;

·(6)Jian Shang, Yushun Wan, Chuming Luo, Gang Ye, Qibin Geng, AshleyAuerbach, Fang Li. Cell entry mechanisms of SARS-CoV-2. Proceedings of the National Academy of Sciences May 2020, 117(21)11727-11734 ;DOI: 10.1073/pnas.2003138117

·(7)Allison J. Greaney, Andrea N. Loes, Katharine H. D. Crawford, Tyler N. Starr, Keara D. Malone, Helen Y. Chu, Jesse D. Bloom, bioRxiv 2020.12.31.425021; doi: https://doi. org/10.1101/2020.12.31.425021

·(8)Nicholas G.Davies, Rosanna C.Barnard, Christopher I.Jarvis, AdamJ.Kucharski, James Munday, Carl A.B.Pearson, Timothy W.Russell, Damien C.Tully, Sam Abbott, Amy Gimma, William Waites, Kerry LM Wong, Kevin van Zandvoort, CMMID COVID-19 Working Group, Rosalind M. Eggo, Sebastian Funk, Mark Jit, Katherine E. Atkins, W. John Edmunds. Estimated transmissibility and severity of novelSARS-CoV-2 Variant of Concern 202012/01 in England.medRxiv2020.12.24.20248822; doi: https://doi.org/10.1101/2020.12.24.20248822

·(9) Houriiyah Tegally, Eduan Wilkinson, Marta Giovanetti, et al. Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2(SARS-CoV-2) lineage with multiple spike mutations in South Africa.medRxiv 2020.12.21.20248640; doi: https://doi.org/10.1101/2020.12.21.20248640

·(10)Kim JS, Jang JH, Kim JM, Chung YS, Yoo CK, Han MG.Genome-WideIdentification and Characterization of Point Mutations in the SARS-CoV-2Genome.Osong Public Health Res Perspect.2020; 11(3) :101-111.doi:10.24171/j.phrp.2020.11.3.05

(11)Nilgiriwala K, Mandal A, Patel G, Mestry T, Vaswani S, Shaikh A, Sriraman K, Parikh S, Udupa S, Chatterjee N, Shastri J, Mistry N. Genome Sequences of Five SARS-CoV-2 Variants from Mumbai , India, Obtained by NanoporeSequencing.Microbiol Resour Announc.2021 Apr15;10(15):e00231-21

·(12)Wenjuan Zhang, Brian D Davis, Stephanie S Chen, Jorge M SincuirMartinez, Jasmine T Plummer, Eric Vail.Emergence of a Novel SARS-CoV-2 Variantin Southern California.JAMA.2021 Apr 6;325(13): 1324-1326

For example, mutation N501Y is a non-synonymous mutation in the receptor binding domain (RBD) of the S protein that is shared by three SARS-CoV-2 lineages, B.1.1.7, P.1 (aka 20J/501Y.V3), and 501Y.V2, first identified in southeast England, Brazil/Japan, and South Africa, respectively. This mutation is one of the key contact residues in the RBD and has been identified to increase binding affinity to human and mouse ACE2. The E484K mutation within the receptor binding domain (RBD) of the S protein (present in new lineages 501Y.S2 and B.1.1.28 from South Africa and Brazil, respectively) affects a residue within the RBD that has been shown to be important for binding to many neutralizing antibodies. The E484Q mutation within the receptor binding domain (RBD) of the S protein (present in new lineages B.1.617 and B.1.429 from India and Denmark, respectively) also affects the same residue within the RBD. Studies have shown that the L452R mutation in lineages B.1.617, B1.427, and B1.429 can stabilize the interaction between the spike protein and its human ACE2 receptor, thereby increasing the infectivity of the virus. Therefore, this mutation affects antibody recognition and enables SARS-CoV-2 to escape immunity. Viruses carrying this mutation have been shown to evade recognition by antibodies in human convalescent sera and can therefore alter the effectiveness of vaccines (see, e.g., Allison J. Greaney, Andrea N. Loes, Katharine H. D. Crawford, Tyler N. Starr, Keara D. Malone, Helen Y. Chu, Jesse D. Bloom, bioRxiv 2020.12.31.425021). Several other mutations have also been found. Mutations K417N, K417T, V367F, N354D, W436R, or V483A, T478K of the S1 protein have been shown to have a higher affinity for binding to ACE2. It has been previously reported that in MERS and SARS-CoV studies, the V483A and G476S mutations were associated with increased affinity for binding to human receptors. On the other hand, R408I may reduce the binding affinity of ACE2. Therefore, according to the present invention, major naturally occurring mutations include K417N mutation in SEQ ID NO: 1 (wherein the amino acid residue (K) at position 417 in SEQ ID NO: 1 is substituted by an amino acid residue (N)), K417T mutation in SEQ ID NO: 1 (wherein the amino acid residue (K) at position 417 in SEQ ID NO: 1 is substituted by an amino acid residue (T)), E484K mutation in SEQ ID NO: 1 (wherein the amino acid residue (E) at position 484 in SEQ ID NO: 1 is substituted by an amino acid residue (K)), E484Q mutation in SEQ ID NO: 1 (wherein the amino acid residue (E) at position 484 is substituted by an amino acid residue (Q)), L452R mutation in SEQ ID NO: 1 (wherein the amino acid residue (L) at position 452 in SEQ ID NO: 1 is substituted by an amino acid residue (R)), T478K mutation in SEQ ID NO: 1 (wherein the amino acid residue (T) at position 478 is substituted by an amino acid residue (K)), and N501Y mutation in NO: 1 (wherein the amino acid residue (N) at position 501 in SEQ ID NO: 1 is substituted with an amino acid residue (Y)).

As used herein, the term "non-naturally occurring mutation" refers to any mutation inserted into the polypeptide of the present invention by genetic engineering. In particular, the mutation is inserted to facilitate the production of the polypeptide. For example, the mutation includes the mutation C538S in SEQ ID NO: 1, wherein the amino acid residue (C) at position 538 in SEQ ID NO: 1 is substituted by an amino acid residue (S). The mutation is particularly useful for avoiding the generation of disulfide bonds in the polypeptide of the present invention. As used herein, the term "encoding" refers to the inherent properties of a specific sequence of nucleotides in a polynucleotide, such as, for example, a gene, cDNA or mRNA used as a template for the synthesis of other polymers and macromolecules in a biological process, which polymers and macromolecules have a defined nucleotide sequence (e.g., rRNA, tRNA and mRNA) or a defined amino acid sequence and the biological properties derived therefrom. Therefore, if the mRNA corresponding to a gene is transcribed and translated in a cell or other biological system to produce a protein, then the gene, cDNA or RNA encodes a protein. Both the coding strand (whose nucleotide sequence is the same as the mRNA sequence, usually provided in the sequence listing) and the non-coding strand (used as a template for transcription of a gene or cDNA) can be referred to as encoding a protein, or other products of the gene or cDNA. Unless otherwise indicated, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns to some extent, so that a nucleotide sequence encoding a protein in a certain version may contain introns.

As used herein, the terms "vector", "cloning vector" and "expression vector" refer to vectors that can introduce DNA or RNA sequences (e.g., foreign genes) into host cells to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequences.

As used herein, the term "promoter/regulatory sequence" refers to a nucleic acid sequence (such as, for example, a DNA sequence) that is recognized by the synthetic machinery of the cell or the synthetic machinery introduced, and the nucleic acid sequence is required to initiate specific transcription of a particular polynucleotide sequence, thereby allowing expression of a gene product that is operably connected to the promoter/regulatory sequence. In some cases, the sequence can be a core promoter sequence, and in other cases, the sequence can also include enhancer sequences and other regulatory elements required for expressing gene products. For example, a promoter/regulatory sequence can be, for example, a sequence that expresses a gene product in a tissue-specific manner.

As used herein, the term "operably linked" or "transcriptional control" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence resulting in the expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functionally related manner with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other, for example, where necessary, two protein coding regions are linked in the same reading frame.

As used herein, the term "transformation" refers to the introduction of "exogenous" (i.e., foreign (extrinsic) or extracellular) genes, DNA or RNA sequences into a host cell, so that the host cell will express the introduced genes or sequences to produce a desired substance, usually a protein or enzyme encoded by the introduced genes or sequences. A host cell that receives and expresses the introduced DNA or RNA is "transformed".

As used herein, the term "expression system" refers to a host cell and a compatible vector, for example, under appropriate conditions for expressing a protein encoded by foreign DNA carried by the vector and introduced into a host cell.

As used herein, the term "spike protein" or "protein S" refers to the SARS-Cov-2 spike glycoprotein that binds to its cell receptor (ie, ACE2) and mediates membrane fusion and viral entry. Each monomer of the trimeric S protein is about 180kDa and contains two subunits, S1 and S2, which mediate attachment and membrane fusion, respectively. Specifically, the spike protein S1 attaches the virus particles to the cell membrane by interacting with the host receptor (ie, human ACE2 receptor) through its "receptor binding domain" (also known as "RBD"). The spike protein S2 mediates the fusion of virus particles and cell membranes by acting as a class I viral fusion protein. Under the current model, the protein has at least three conformational states: the native state before fusion, the intermediate state before the hairpin, and the hairpin state after fusion. In the process of fusion between the virus and the target cell membrane, the coiled coil region (heptad repeat) presents a trimer structure of a hairpin, positioning the fusion peptide close to the C-terminal region of the extracellular domain. The formation of this structure seems to promote the apposition and subsequent fusion of the virus and target cell membranes. The spike protein S2' acts as a viral fusion peptide, which is exposed (unmasked) after S2 cleavage during viral endocytosis. Generally, the spike protein has an amino acid sequence as shown in SEQ ID NO:1.

SEQ ID NO: 1>sp|P0DTC2|Spike protein_SARS2 spike glycoprotein OS=Severe acute respiratory syndrome coronavirus 2 OX=2697049GN=S PE=1SV=1. The RBD sequence is underlined.

As used herein, the term "RBD polypeptide" refers to a polypeptide consisting of an amino acid sequence that is at least 90% identical to an amino acid sequence ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO:1.

In some embodiments, the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO:1.

In some embodiments, the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1, and comprising one or more non-naturally occurring mutations. In some embodiments, the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1, and comprising a non-natural mutation at position 538. In some embodiments, the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1, and comprising a C538S mutation.

In some embodiments, the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1, and comprising one or more naturally occurring mutations. In some embodiments, the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1, and comprising one or more naturally occurring mutations at positions 417, 452, 478, 484, or 501. In some embodiments, the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1, and comprising one or more naturally occurring mutations in positions selected from K417N, K417T, E484K, and N501Y mutations. In some embodiments, the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO:1 and comprising one or more naturally occurring mutations in a position selected from K417N, K417T, L452R, T478K, E484Q, E484K and N501Y mutations.

In some embodiments, the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1 and comprising a N501Y naturally occurring mutation and a C538S non-naturally occurring mutation.

In some embodiments, the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1, and comprising naturally occurring mutations of K417T, E484K, N501Y and a non-naturally occurring mutation of C538S.

In some embodiments, the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1, and comprising naturally occurring mutations of K417N, E484K, N501Y and a non-naturally occurring mutation of C538S.

In some embodiments, the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1, and comprising naturally occurring mutations K417T, E484Q, N501Y and a non-naturally occurring mutation C538S.

In some embodiments, the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1, and comprising naturally occurring mutations of K417N, E484Q, N501Y and a non-naturally occurring C538S mutation.

In some embodiments, the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1, and comprising naturally occurring mutations of K417N, T478K, E484Q, L452R and N501Y and a non-naturally occurring C538S mutation.

In some embodiments, the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1, and comprising naturally occurring mutations of K417N, T478K, E484K, L452R and N501Y and a non-naturally occurring C538S mutation.

As used herein, the term "conjugate" or interchangeably "conjugated polypeptide" is intended to mean a combination or chimeric molecule formed by covalently linking one or more polypeptides. The term "covalently linked" or "conjugated" means that the polypeptide and the non-peptide portion are covalently linked to each other directly, or indirectly through one or more spacer moieties (e.g., a bridge, a spacer, or one or more bond moieties). A specific conjugate is a fusion protein.

As used herein, the term "fusion protein" refers to a protein produced by connecting two or more polypeptides derived from isolated proteins. Specifically, fusion proteins can be produced by recombinant DNA technology and are generally used in biological research or treatment. Fusion proteins can also be produced by chemical covalent coupling, whether with or without a linker between the polypeptide portions of the fusion protein. In a fusion protein, two or more polypeptides are fused directly or through a linker.

As used herein, the term "directly" refers to the first amino acid at the N-terminus of the first polypeptide being fused to the last amino acid at the C-terminus of the second polypeptide. This direct fusion can occur naturally, and descriptions are found in (Vigneron et al., Science 2004, PMID 15001714), (Warren et al., Science 2006, PMID 16960008), (Berkers et al., J. Immunol. 2015a, PMID 26401000), (Berkers et al., J. Immunol. 2015b, PMID 26401003), (Delong et al., Science 2016, PMID 26912858), (Liepe et al., Science 2016, PMID 27846572), (Babon et al., Nat. Med. 2016, PMID 27798614).

As described herein, the term "linker" has a general meaning in the art and refers to an amino acid sequence of sufficient length to ensure that the protein forms a suitable secondary and tertiary structure. In some embodiments, the linker is a peptide linker comprising at least one, but less than 30 amino acids, for example, 2-30 amino acids, preferably 10-30 amino acids, more preferably 15-30 amino acids, more preferably 19-27 amino acids, and most preferably 20-26 amino acids. In some embodiments, the linker has 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30 amino acid residues. Typically, the linker is a linker that allows the compound to adopt an appropriate conformation (i.e., the conformation allows the IL-15Rβ/γ signaling pathway to have appropriate signal transduction activity). The most suitable linker sequences (1) will adopt a flexible, extended conformation, (2) will not exhibit a tendency to form ordered secondary structures that could interact with the functional domains of the fusion protein, and (3) will have minimal hydrophobic or charged characteristics that can facilitate interaction with the functional protein domains.

As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, i.e., molecules containing antigen binding sites that immunospecifically bind to antigens. In natural antibodies of rodents and primates, two heavy chains are connected to each other by disulfide bonds, and each heavy chain is connected to a light chain by a disulfide bond. There are two types of light chains, λ (1) and κ (k). There are five main heavy chain categories (or isotypes), which determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA and IgE. Each chain contains different sequence domains. In a typical IgG antibody, the light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity to the antigen. The constant region domains of the light chain (CL) and heavy chain (CH) confer important biological properties such as antibody chain attachment, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcRs). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portion of one light chain and one heavy chain. The specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site consists of residues primarily from the hypervariable regions or complementary determining regions (CDRs). Sometimes, residues from non-hypervariable regions or framework regions (FRs) can participate in the antibody binding site or affect the overall domain structure and thus the binding site. The complementary determining regions or CDRs refer to the amino acid sequences that together define the binding affinity and specificity of the native Fv region of the natural immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, referred to as L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3. Therefore, the antigen binding site usually includes six CDRs, including a CDR group from each of the heavy chain and light chain V regions. Framework region (FR) refers to the amino acid sequence between CDRs. Therefore, the variable region of the light chain and heavy chain usually contains 4 framework regions and 3 CDRs in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The residues in the antibody variable domain are usually numbered according to the system designed by Kabat et al. This system is shown in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, NIH, USA (Kabat et al., 1992, hereinafter referred to as "Kabat et al."). Kabat residue names do not always directly correspond to the linear numbering of amino acid residues in the SEQ ID sequence. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering, corresponding to shortened structural components or inserted structural components in the basic variable domain structure (whether framework or complementary determining region (CDR)). For a given antibody, the correct Kabat residue numbering can be determined by aligning the homologous residues in the antibody sequence with the "standard" Kabat numbering sequence. According to the Kabat numbering system, the CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2), and residues 95-102 (H-CDR3). According to the Kabat numbering system, the CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2), and residues 89-97 (L-CDR3). For the agonist antibodies described below, the CDRs have been determined using the CDR search algorithm from www.bioinf.org.uk - see the section entitled "How to identify the CDRs by looking at a sequence" on the antibody page.

As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (such as, for example, a heavy chain antibody chain or a light chain), or to a polypeptide consisting essentially of such a globular region.

As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. The human IgG heavy chain Fc region is generally defined as comprising the amino acid residues from position C226 or from position P230 to the carboxyl terminus of an IgG antibody. The residue numbering in the Fc region is the EU index of Kabat. The C-terminal lysine (residue K447) in the Fc region can be removed, for example, during the production or purification of the antibody. Therefore, the antibody composition of the present invention can include antibody groups having all K447 residues removed, antibody groups having the K447 residue not removed, and antibody groups having an antibody mixture containing or not containing the K447 residue.

As used herein, the term "chimeric antibody" refers to an antibody comprising a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody. In one embodiment, a "chimeric antibody" is an antibody molecule in which (a) the constant region (i.e., heavy chain and/or light chain) or a portion thereof is altered, replaced or exchanged so that the antigen binding site (variable region) is connected to a constant region of a different or altered class, effector function and/or type, or a completely different molecule (e.g., enzyme, toxin) that imparts new properties to chimeric antibodies of agonist molecules (e.g., CD40 ligands, hormones, growth factors, drugs, etc.); or (b) the variable region or a portion thereof is altered, replaced or exchanged by a variable region with a different or altered antigen specificity. Chimeric antibodies also include primatized antibodies, particularly humanized antibodies. In addition, chimeric antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. For details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). (See U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

As used herein, the term "humanized antibody" includes antibodies having 6 CDRs of a mouse antibody but with humanized framework and constant regions. More specifically, the term "humanized antibody" used herein may include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted to human framework sequences.

As used herein, the term "human monoclonal antibody" is intended to include antibodies having variable regions and constant regions derived from human immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo). However, in one embodiment, as used herein, the term "human monoclonal antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been transplanted to human framework sequences.

As used herein, the term "immune response" refers to the response of the immune system to an antigen in the host, including the production of antigen-specific antibodies and/or cytotoxic responses. For the immune response to the initial antigen exposure (primary immune response), it can usually be detected after a lag period of several days to two weeks; the immune response to subsequent stimulation of the same antigen (secondary immune response) is faster than the primary immune response. The immune response to the transgenic product can include humoral (e.g., antibody response) and cellular (e.g., cytolytic T cell response) immune responses, which can be caused by the immunogenic product encoded by the transgene. The level of the immune response can be measured by methods known in the art (e.g., by measuring antibody titers).

As used herein, the term "APC" or "antigen presenting cell" refers to a cell capable of activating T cells, including but not limited to certain macrophages, B cells, and dendritic cells.

As used herein, the term "dendritic cell" or "DC" refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their unique morphology, high levels of surface expression of MHC class II (Steinman et al., Ann Rev. Immunol. 9:271 (1991); the description of such cells is incorporated herein by reference).

As used herein, the term "CD40" has its ordinary meaning in the art and refers to the human CD40 polypeptide receptor. In some embodiments, CD40 is an isoform of the human canonical sequence reported by UniProtKB-P25942 (also known as human TNR5).

As used herein, the term "CD40L" has its ordinary meaning in the art and refers to a human CD40L polypeptide, for example, as reported in UniProtKB-P25942, including its CD40 binding domain of SEQ ID NO: 47. CD40L can be expressed as a soluble polypeptide and is a natural ligand for the CD40 receptor.

SEQ ID NO:47>CD40L binding domain

As used herein, the term "CD40 agonist antibody" refers to an antibody that increases CD40-mediated signaling activity in a cell-based assay (such as a B cell proliferation assay) in the absence of CD40L. In particular, the CD40 agonist antibody (i) induces B cell proliferation, as measured in vitro by flow cytometry analysis, or by repeated dilution analysis of CFSE-labeled cells; and/or (ii) induces the secretion of cytokines (such as IL-6, IL-12 or IL-15), as measured in vitro using a dendritic cell activation assay.

As used herein, the term "Langerin" has its ordinary meaning in the art and refers to a human C-type lectin domain family 4 member K polypeptide. In some embodiments, Langerhansin is an isoform of the human canonical sequence reported by UniProtKB-Q9UJ71 (also known as human CD207).

As used herein, the term "treatment" or "treat" refers to prophylactic or preventive treatment, as well as curative or disease-modifying treatment, including treatment of patients at risk of contracting a disease or suspected of contracting a disease, and patients who are ill or diagnosed with a disease or medical condition, including suppression of clinical relapse. Treatment can be applied to patients with a medical condition or who may eventually acquire a condition to prevent, cure, delay the onset of the condition, reduce the severity of the condition, or improve one or more symptoms of the condition or recurrence of the condition, or to extend the patient's survival beyond the expected survival without such treatment. "Therapeutic regimen" refers to a treatment pattern for a disease, such as a dosing pattern used during treatment. The therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction phase" refers to a therapeutic regimen (or part of a therapeutic regimen) used for the initial treatment of a disease. The overall goal of an induction regimen is to provide patients with high levels of drug in the initial stages of a therapeutic regimen. The induction regimen may employ (in part or in whole) a "loading regimen," which may include administering a larger dose of the drug than the physician would administer during the maintenance regimen, administering the drug more frequently than the physician would administer during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance phase" refers to a treatment regimen (or portion of a treatment regimen) used to maintain a patient during treatment of a disease, e.g., to keep the patient in remission for a long period of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administration of the drug at regular intervals, e.g., weekly, monthly, yearly, etc.), or intermittent therapy (e.g., interruption of treatment, intermittent treatment, treatment upon relapse, or treatment when specific predetermined criteria are met [e.g., pain, disease manifestations, etc.]).

As used herein, the term "pharmaceutical composition" refers to a composition as described herein or a pharmaceutically acceptable salt thereof, and other agents, such as carriers and/or excipients. The pharmaceutical compositions provided herein generally include a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. suitable for the particular dosage form desired. Remington's Pharmaceutical-Sciences, 16th edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers for formulating pharmaceutical compositions and known techniques for preparing the same.

As used herein, the term "vaccination" or "vaccinating" refers to, but is not limited to, the process of eliciting an immune response against a specific antigen in a subject.

As used herein, the term "vaccine composition" refers to a composition that can be administered to humans or animals to induce an immune system response; such an immune system response can result in the activation of certain cells, particularly APCs, T lymphocytes, and B lymphocytes.

As used herein, the term "antigen" refers to a molecule that can be specifically bound by an antibody or T cell receptor (TCR) if processed and presented by an MHC molecule. Antigens can also be recognized by the immune system and/or can induce a humoral immune response and/or a cellular immune response, resulting in activation of B and/or T lymphocytes. Antigens can have one or more epitopes or antigenic sites (B and T epitopes).

As used herein, the term "adjuvant" refers to a compound that can induce and/or enhance an immune response to an antigen when administered to a subject or animal. Adjuvant is also intended to mean a substance whose action is generally of the nature of accelerating, prolonging or enhancing a specific immune response to a specific antigen. In the context of the present invention, the term "adjuvant" refers to a compound that enhances the innate immune response by affecting the transient response of the innate immune response, and enhances the longer-term effects of the adaptive immune response by the activation and maturation of antigen presenting cells (APCs), especially dendritic cells (DCs).

As used herein, the expression "therapeutically effective amount" refers to a sufficient amount of the active ingredient of the present invention to induce an immune response at a reasonable benefit/risk ratio applicable to medical treatment.

As used herein, the term "immune checkpoint inhibitor" has its general meaning in the art, referring to any compound that inhibits the function of immunosuppressive checkpoint proteins. As used herein, the term "immune checkpoint protein" has its general meaning in the art, referring to molecules expressed by T cells, which turn on signals (stimulatory checkpoint molecules) or turn off signals (inhibitory checkpoint molecules). Immune checkpoint molecules are considered to constitute immune checkpoint pathways similar to CTLA-4 and PD-1 dependent pathways in the art (see, e.g., Pardoll, 2012.Nature Rev Cancer 12: 252-264; Mellman et al., 2011.Nature 480: 480-489). The example of inhibitory checkpoint molecules includes A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA.

Antibodies of the present invention

The first object of the present invention relates to antibodies directed against surface antigens of antigen-presenting cells, wherein the heavy chain and/or light chain is coupled or fused to an RBD polypeptide.

In some embodiments, the heavy chain of the antibody is coupled or fused to the RBD polypeptide.

In some embodiments, the light chain of the antibody is coupled or fused to the RBD polypeptide.

In some embodiments, the heavy and light chains of the antibody are coupled or fused to the RBD polypeptide.

In some embodiments, the heavy chain is fused or coupled to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO:1 and comprising the C538S non-naturally occurring mutation, and the light chain is coupled or fused to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO:1 and comprising the K417N, E484K, N501Y naturally occurring mutations and the C538S non-naturally occurring mutation.

In some embodiments, the light chain is fused or coupled to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO:1 and comprising the non-naturally occurring mutation C538S, and the heavy chain is coupled or fused to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO:1 and comprising the naturally occurring mutations K417N, E484K, N501Y and the non-naturally occurring mutation C538S.

In some embodiments, the heavy chain is fused or coupled to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO:1 and comprising a non-naturally occurring mutation of C538S, and the light chain is coupled or fused to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO:1 and comprising naturally occurring mutations of K417N, E484Q, N501Y and a non-naturally occurring mutation of C538S.

In some embodiments, the light chain is fused or coupled to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO:1 and comprising the non-naturally occurring mutation C538S, and the heavy chain is coupled or fused to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO:1 and comprising the naturally occurring mutations K417N, E484Q, N501Y and the non-naturally occurring mutation C538S.

In some embodiments, the heavy chain is fused or coupled to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 of SEQ ID NO:1 and comprising the C538S non-naturally occurring mutation, and the light chain is coupled or fused to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 of SEQ ID NO:1 and comprising K417N, L452R, T478K, E484Q, N501Y naturally occurring mutations and C538S non-naturally occurring mutation.

In some embodiments, the light chain is fused or coupled to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO:1 and comprising the C538S non-naturally occurring mutation, and the heavy chain is coupled or fused to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO:1 and comprising K417N, L452R, T478K, E484Q, N501Y naturally occurring mutations and C538S non-naturally occurring mutation.

In some embodiments, the heavy chain is fused or coupled to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 of SEQ ID NO:1 and comprising the C538S non-naturally occurring mutation, and the light chain is coupled or fused to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 of SEQ ID NO:1 and comprising K417N, L452R, T478K, E484K, N501Y naturally occurring mutations and C538S non-naturally occurring mutation.

In some embodiments, the light chain is fused or coupled to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 of SEQ ID NO:1 and comprising the C538S non-naturally occurring mutation, and the heavy chain is coupled or fused to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 to amino acid residue 541 of SEQ ID NO:1 and comprising K417N, L452R, T478K, E484K, N501Y naturally occurring mutations and C538S non-naturally occurring mutation.

In some embodiments, the antibody is an IgG antibody, preferably an IgG1 or IgG4 antibody, or even more preferably an IgG4 antibody.

In some embodiments, the antibody is a chimeric antibody, particularly a chimeric mouse/human antibody.

In some embodiments, the antibody is a humanized antibody.

Chimeric antibodies or humanized antibodies can be prepared based on the sequence of the mouse monoclonal antibody prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, in order to produce chimeric antibodies, mouse variable regions can be connected to human constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567, Cabilly et al.). In order to produce humanized antibodies, mouse CDR regions can be inserted into human frameworks using methods known in the art. See, e.g., U.S. Patent No. 5,225,539 of Winter and U.S. Patent Nos. 5,530,101 of Queen et al.; 5,585,089; 5,693,762 and 6,180,370.

In some embodiments, the antibodies are human antibodies. In some embodiments, transgenic or transchromosomal mice carrying parts of the human immune system other than the mouse system can be used to identify human antibodies. These transgenic and transchromosomal mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "human Ig mice". HuMAb (Medarex, Inc.) contains human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, as well as targeted mutations that inactivate endogenous μ and κ chain loci (see, e.g., Lonberg et al., 1994 Nature 368 (6474): 856-859). In another embodiment, mice carrying human immunoglobulin sequences on transgenes and transchromosomes, such as mice carrying human heavy chain transgenes and human light chain transchromosomes, can be used to produce human anti-PD-1 antibodies. Such mice are referred to herein as "KM mice" and are described in detail in PCT Publication WO 02/43478 by Ishida et al.

In some embodiments, the antibody is specific for a cell surface marker of a professional APC. The antibody may be specific for a cell surface marker of another professional APC, such as a B cell or a macrophage.

In some embodiments, the antibody is selected from an antibody that specifically binds to DC immune receptor (DCIR), MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerhansin, DECTIN-1, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1, Fey receptor, LOX-1, and ASPGR.

In some embodiments, the antibody is specific for CD40.

In some embodiments, the anti-CD40 antibody is derived from the 12E12 antibody and comprises:

- a heavy chain comprising complementary determining regions CDR1H, CDR2H and CDR3H, wherein the CDR1H has the amino acid sequence GFTFSDYYMY (SEQ ID NO: 2), the CDR2H has the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID NO: 3) and the CDR3H has the amino acid sequence RGLPFHAMDY (SEQ ID NO: 4),

-–and a light chain comprising complementary determining regions CDR1L, CDR2L and CDR3L, wherein CDR1L has the amino acid sequence SASQGISNYLN (SEQ ID NO:5), the CDR2L has the amino acid sequence YTSILHS (SEQ ID NO:6) and the CDR3L has the amino acid sequence QQFNKLPPT (SEQ ID NO:7).

In some embodiments, the anti-CD40 antibody is derived from the 11B6 antibody and comprises:

- a heavy chain comprising complementary determining regions CDR1H, CDR2H and CDR3H, wherein CDR1H has the amino acid sequence GYSFTGYYMH (SEQ ID NO: 8), CDR2H has the amino acid sequence RINPYNGATSYNQNFKD (SEQ ID NO: 9) and CDR3H has the amino acid sequence EDYVY (SEQ ID NO: 10), and

-–a light chain comprising complementary determining regions CDR1L, CDR2L and CDR3L, wherein CDR1L has the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO: 11), the CDR2L has the amino acid sequence KVSNRFS (SEQ ID NO: 12) and the CDR3L has the amino acid sequence SQSTHVPWT (SEQ ID NO: 13).

In some embodiments, the anti-CD40 antibody is derived from the 12B4 antibody and comprises:

a heavy chain comprising complementary determining regions CDR1H, CDR2H and CDR3H, wherein CDR1H has the amino acid sequence GYTFTDYVLH (SEQ ID NO: 14), CDR2H has the amino acid sequence YINPYNDGTKYNEKFKG (SEQ ID NO: 15), and CDR3H has the amino acid sequence GYPAYSGYAMDY (SEQ ID NO: 16), and

-- A light chain comprising complementary determining regions CDR1L, CDR2L and CDR3L, wherein CDR1L has the amino acid sequence RASQDISNYLN (SEQ ID NO: 17), the CDR2L has the amino acid sequence YTSRLHS (SEQ ID NO: 18) and the CDR3L has the amino acid sequence HHGNTLPWT (SEQ ID NO: 19).

In some embodiments, the anti-CD40 antibody is selected from the group consisting of mAb1, mAb2, mAb3, mAb4, mAb5, and mAb6 selected as described in Table A.

Table A: CD40 Antibodies

SEQ ID NO: 20 (Amino acid sequence of the variable heavy chain region (VH) (v2) of humanized 11B6)

SEQ ID NO: 21 (amino acid sequence of the variable light chain region (VL) Vk (v2) of humanized 11B6 VL)

SEQ ID NO: 22 (amino acid sequence of the variable heavy chain region VH (v3) of humanized 11B6)

SEQ ID NO:23 (VH amino acid sequence of mAb3 (12B4))

SEQ ID NO:24 (VL amino acid sequence of mAb3 (12B4))

SEQ ID NO:25 (VH amino acid sequence of mAb4 (24A3 HC))

SEQ ID NO:26 (VL amino acid sequence of mAb4 (24A3 KC))

SEQ ID NO:27 (VH amino acid sequence of mAb5)

SEQ ID NO:28 (VL amino acid sequence of mAb5)

SEQ ID NO:29 (VH amino acid sequence of mAb6 (12E12 H3 humanized HC))

SEQ ID NO:30 (VL amino acid sequence of mAb6 (humanized K2 12E12))

In some embodiments, the anti-CD40 antibody is a CD40 agonist antibody. CD40 agonist antibodies are described in WO2010/009346, WO2010/104747, and WO2010/104749. Other anti-CD40 agonist antibodies under development include CP-870,893, which is a fully human IgG2 CD40 agonist antibody developed by Pfizer. It binds to CD40 with a KD of 3.48×10-10M, but does not block the binding of CD40L (see, e.g., U.S. Pat. No. 7,338,660) and SGN-40, which is a humanized IgG1 antibody developed by Seattle Genetics from mouse antibody clone S2C6, which was generated using a human bladder cancer cell line as an immunogen. SGN-40 binds to CD40 with a KD of 1.0×10-9 M and exerts its effect by enhancing the interaction between CD40 and CD40L, thereby exhibiting a partial agonist effect (Francisco J A, et al., Cancer Res, 60: 3225-31, 2000). Even more particularly, the CD40 agonist antibody is selected from mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6 selected as described in Table A.

In some embodiments, the heavy chain or light chain (ie, the chain not coupled or fused to the RBD polypeptide) of a CD40 agonist antibody is coupled or fused to the CD40 binding domain of CD40L.

In some embodiments, the CD40 binding domain of CD40L is fused to the C-terminus of the light chain or heavy chain of the CD40 agonist antibody, optionally via a linker, preferably a FlexV1 linker as described below.

In some embodiments, the antibodies of the invention consist of a CD40 agonist antibody, wherein the heavy chain of the antibody is fused or coupled to the RBD polypeptide and the light chain is coupled or fused to the CD40 binding domain of CD40L (SEQ ID NO: 47).

In some embodiments, the antibody is specific for Langerhansin. In some embodiments, the antibody is derived from antibody 15B10 with ATCC accession number PTA-9852. In some embodiments, the antibody is derived from antibody 2G3 with ATCC accession number PTA-9853. In some embodiments, the antibody is derived from antibodies 91E7, 37C1 or 4C7 as described in WO2011032161.

In some embodiments, the anti-Langerhansin antibody comprises a heavy chain comprising the complementarity determining regions CDR1H, CDR2H, and CDR3H of the 15B10 antibody, and a light chain comprising the complementarity determining regions CDR1L, CDR2L, and CDR3L of the 15B10 antibody.

In some embodiments, the anti-Langerhansin antibody comprises a heavy chain containing the complementarity determining regions CDR1H, CDR2H, and CDR3H of the 2G3 antibody, and a light chain containing the complementarity determining regions CDR1L, CDR2L, and CDR3L of the 2G3 antibody.

In some embodiments, the anti-Langerhansin antibody comprises a heavy chain containing the complementarity determining regions CDR1H, CDR2H, and CDR3H of the 4C7 antibody, and a light chain containing the complementarity determining regions CDR1L, CDR2L, and CDR3L of the 4C7 antibody.

In some embodiments, the antibody is selected from mAb7, mAb8, mAb9, mAb10, mAb11, and mAb12 selected as described in Table B.

SEQ ID NO:31 (amino acid sequence of the variable heavy chain region (VH) of 15B10)

SEQ ID NO:32 (amino acid sequence of the variable light chain (VL) of 15B10)

SEQ ID NO: 33 (amino acid sequence of the variable heavy chain region (VH) of 2G3)

SEQ ID NO:34 (amino acid sequence of variable light chain (VL) of 2G3)

SEQ ID NO:35 (amino acid sequence of the heavy chain of 4C7)

SEQ ID NO:36 (amino acid sequence of the light chain of 4C7)

The antibodies of the present invention can be produced by any technique known in the art, such as, but not limited to, any chemical, biological, genetic or enzymatic technique (used alone or in combination). Knowing the amino acid sequence of the desired sequence, those skilled in the art can easily produce the polypeptide by standard techniques for producing polypeptides. For example, the antibodies of the present invention can be synthesized by recombinant DNA techniques now known in the art. For example, the DNA sequence encoding the desired (poly)peptide is incorporated into an expression vector, and after such a vector is introduced into a suitable eukaryotic or prokaryotic host that will express the desired polypeptide, these fragments can be obtained as DNA expression products, and then these fragments can be separated from the host using known techniques.

The heavy chain and/or light chain of the antibody is coupled or fused to the RBD polypeptide via its C-terminus. In some embodiments, the heavy chain and/or light chain of the antibody is fused to the N-terminus of the RBD polypeptide.

In some embodiments, the heavy chain and/or light chain of the antibody is coupled to the RBD polypeptide by using chemical coupling. Several methods for connecting or coupling the antibody to its conjugate portion are known in the art. Examples of the types of joints that have been used to couple part to the antibody include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing joints, such as valine-citrulline joints. It can be selected, for example, a joint that is susceptible to low pH cutting in the lysosomal compartment or susceptible to protease (e.g., a protease preferably expressed in tumor tissue, such as cathepsin (cathepsin) (e.g., cathepsin B, C, D)) cutting. Techniques for coupling polypeptides, in particular, are well known in the art (see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al., ed., Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (Robinson et al., ed., Marcel Deiker, Inc. 2nd ed., 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al., ed., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibodies In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al., ed., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62: 119-58; see also, e.g., PCT Publication WO 89/12624). Typically, the peptide is covalently linked to a lysine or cysteine residue on the antibody via an N-hydroxysuccinimide ester or maleimide functional group, respectively. It has been reported that the use of engineered cysteine or the incorporation of unnatural amino acids in conjugation methods can improve the homogeneity of the conjugates (Axup, J.Y., Bajjuri, K.M., Ritland, M., Hutchins, B.M., Kim, C.H., Kazane, S.A., Halder, R., Forsyth, J.S., Santidrian, A.F., Stafin, K., et al. (2012). Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 109, 16101-16106.; Junutula, J.R., Flagella, K.M., Graham, R.A., Parsons, K.L., Ha, E., Raab, H., Bhakta, S., Nguyen, T., Dugger, D.L., Li, G. et al. (2010). Engineered thio-trastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin. Cancer Res. 16, 4769-4778). Junutula et al. (Nat Biotechnol. 2008; 26: 925-32) developed a cysteine-based site-specific conjugation called "THIOMAB" (TDC), which is said to have an improved therapeutic index compared to conventional conjugation methods. For ADCs, coupling with non-natural amino acids already incorporated into the antibody is also being explored; however, the generality of this approach has not yet been determined (Axup et al., 2012). In particular, one skilled in the art can also envision engineering Fc-containing polypeptides using tags containing acyl donor glutamine (e.g., peptide tags containing Gin or Q tags) or using endogenous glutamine that is made reactive by polypeptide engineering (e.g., by amino acid deletion, insertion, substitution or mutation on the polypeptide). The transglutaminase can then be covalently cross-linked with an amine donor reagent (e.g., a small molecule containing a reactive amine or attached to a reactive amine) to form a stable and homogeneous population of engineered Fc-containing polypeptide conjugates, wherein the amine donor reagent is site-specifically coupled to the Fc-containing polypeptide (via a tag containing acyl donor glutamine, or accessible/exposed/reactive endogenous glutamine) (WO 2012059882).

In some embodiments, the heavy and/or light chains of the antibody are coupled to the RBD polypeptide via a dockerin domain or multiple domains that allow non-covalent coupling to an adhesion fusion protein (as described in US20160031988A1 and US20120039916A1).

In some embodiments, the heavy chain and/or light chain of the antibody is fused to the RBD polypeptide to form a fusion protein. In some embodiments, the RBD polypeptide is fused directly to or through a linker to the heavy chain or light chain. As used herein, the term "directly" refers to the first amino acid at the N-terminus of the RBD polypeptide being fused to the last amino acid at the C-terminus of the heavy chain or light chain. Such direct fusions may occur naturally and are described in (Vigneron et al., Science 2004, PMID 15001714), (Warren et al., Science 2006, PMID 16960008), (Berkers et al., J. Immunol. 2015a, PMID 26401000), (Berkers et al., J. Immunol. 2015b, PMID 26401003), (Delong et al., Science 2016, PMID 26912858), (Liepe et al., Science 2016, PMID 27846572), (Babon et al., Nat. Med. 2016, PMID 27798614).

In some embodiments, the linker is selected from FlexV1, f1, f2, f3, or f4 as described below.

QTPTNTISVTPTNNSTPTNNSNPKPNP(flexV1, SEQ ID NO: 37)

SSVSPTTSVHPTPTSVPPTPTKSSP(f1, SEQ ID NO: 38)

PTSTPADSSTITPTATPTATPTIKG(f2, SEQ ID NO: 39)

TVTPTATATPSAIVTTITPTATTKP (f3, SEQ ID NO: 40)

TNGSITVAATAPTVTPTVNATPSAA (f4, SEQ ID NO: 41)

In some embodiments, the antibody comprises i) a heavy chain fused to an RBD polypeptide to form a fusion protein as set forth in SEQ ID NO:42, and ii) a light chain as set forth in SEQ ID NO:43.

SEQ ID NO:42 (h anti-CD40 VH3-LV-hIgG4H-C-AS-virus SARS-CoV-2-spike protein-RBDC221S)

SEQ ID NO:43 (h anti-CD40VK2-LV-hIgGK-C)

In some embodiments, the antibody comprises i) a heavy chain fused to an RBD polypeptide to form a fusion protein as set forth in SEQ ID NO:42, and ii) a light chain fused to an RBD polypeptide to form a fusion protein as set forth in SEQ ID NO:44.

SEQ ID NO:44 [h anti-CD40 VK2-LV-hIgGK-C-virus SARS-CoV-2-spike protein-RBDC221SSA variant]

In some embodiments, the antibody comprises i) a heavy chain fused to an RBD polypeptide to form a fusion protein as shown in SEQ ID NO:45, and ii) a light chain fused to an RBD polypeptide to form a fusion protein as shown in SEQ ID NO:46.

SEQ ID NO:45 [h anti-CD40 VH3-LV-hIgGK-C-viral SARS-CoV-2-spike protein-RBDC221SSA variant]

SEQ ID NO:46 [h anti-CD40 VK2-LV-hIgG4H-C-virus SARS-CoV-2-spike protein-RBDC221S]

In some embodiments, the antibody comprises i) a heavy chain fused to an RBD polypeptide to form a fusion protein as set forth in SEQ ID NO:42, and ii) a light chain fused to an RBD polypeptide to form a fusion protein as set forth in SEQ ID NO:48.

SEQ ID NO:48 [h anti-CD40 VK2-LV-hIgGK-C-virus SARS-CoV-2-spike protein-RBDC221S (K417N, L452R, T478K, E484Q, N501Y)]

In some embodiments, the antibody comprises i) a heavy chain fused to an RBD polypeptide to form a fusion protein as set forth in SEQ ID NO:49, and ii) a light chain fused to an RBD polypeptide to form a fusion protein as set forth in SEQ ID NO:46.

SEQ ID NO:49 [h anti-CD40 VH3-LV-hIgGK-C-virus SARS-CoV-2-spike protein-RBDC221S (K417N, L452R, T478K, E484Q, N501Y)]

In some embodiments, the antibody comprises i) a heavy chain fused to an RBD polypeptide to form a fusion protein as shown in SEQ ID NO:42, and ii) a light chain fused to an RBD polypeptide to form a fusion protein as shown in SEQ ID NO:50.

SEQ ID NO:50[h anti-CD40 VK2-LV-hIgGK-C-virus SARS-CoV-2-spike protein-RBDC221S (K417N, L452R, T478K, E484K, N501Y)]

In some embodiments, the antibody comprises i) a heavy chain fused to an RBD polypeptide to form a fusion protein as shown in SEQ ID NO:51, and ii) a light chain fused to an RBD polypeptide to form a fusion protein as shown in SEQ ID NO:46.

SEQ ID NO:51 [h anti-CD40 VH3-LV-hIgGK-C-virus SARS-CoV-2-spike protein-RBDC221S (K417N, L452R, T478K, E484K, N501Y)]

In some embodiments, the antibody comprises i) a heavy chain fused to an RBD polypeptide to form a fusion protein as shown in SEQ ID NO:42, and ii) a light chain fused to an RBD polypeptide to form a fusion protein as shown in SEQ ID NO:52.

SEQ ID NO:52 [h anti-CD40 VK2-LV-hIgGK-C-virus SARS-CoV-2-spike protein-RBDC221S (K417N, E484Q, N501Y)]

In some embodiments, the antibody comprises i) a heavy chain fused to an RBD polypeptide to form a fusion protein as shown in SEQ ID NO:53, and ii) a light chain fused to an RBD polypeptide to form a fusion protein as shown in SEQ ID NO:46.

SEQ ID NO:53 [h anti-CD40 VH3-LV-hIgGK-C-virus SARS-CoV-2-spike protein-RBDC221S (K417N, E484Q, N501Y)]

Nucleic acids, vectors and host cells of the present invention:

Another object of the present invention relates to a nucleic acid encoding a heavy chain and/or light chain of an antibody against a surface antigen of an antigen presenting cell fused to an RBD polypeptide.

Typically, the nucleic acid is a DNA or RNA molecule, which may be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

Therefore, another object of the present invention relates to a vector comprising the nucleic acid according to the invention.

Such vectors may include regulatory elements, such as promoters, enhancers, terminators, etc., to cause or directly express the antibody when the antibody is administered to a subject. Examples of promoters and enhancers for animal cell expression vectors include early promoters and enhancers of SV40, LTR promoters and enhancers of Moloney murine leukemia virus, promoters and enhancers of immunoglobulin H chains, etc. Any animal cell expression vector may be used, as long as the gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSG1βd2-4, etc. Other examples of plasmids include replication plasmids or integrated plasmids containing an origin of replication, such as, for example, pUC, pcDNA, pBR, etc. Other examples of viral vectors include adenovirus, retrovirus, herpes virus, and AAV vectors. This recombinant virus can be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-deficient recombinant viruses can be found, for example, in WO 95/14785, WO 96/22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056, and WO 94/19478.

Another object of the present invention relates to a host cell which has been transfected, infected or transformed with a nucleic acid and/or a vector according to the invention.

The nucleic acid of the present invention can be used to produce the antibody of the present invention in a suitable expression system. Common expression systems include Escherichia coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include Escherichia coli, Kluyveromyces or Saccharomyces yeast. Mammalian host cells include Chinese hamster ovary cells (CHO cells), including dhfr-CHO cells (described in Urlaub and Chasin, 1980) using a DHFR selectable marker, CHOK1 dhfr+ cell lines, NSO myeloma cells, COS cells and SP2 cells (e.g., GS CHO cell lines with GSXceed ^™ gene expression system (Lonza)) or HEK cells.

The present invention also relates to a method for producing a recombinant host cell expressing a polypeptide according to the present invention, the method comprising the steps of: (i) introducing the above-mentioned recombinant nucleic acid or vector into a competent host cell in vitro or in vitro, (ii) culturing the obtained recombinant host cell in vitro or in vitro, and (iii) optionally, selecting cells that express and/or secrete the antibody. Such a recombinant host cell can be used to produce the antibody of the present invention.

Therefore, the host cells disclosed herein are particularly suitable for producing the antibodies of the present invention. Indeed, when recombinant expression is introduced into mammalian host cells, the host cells are cultured to produce the polypeptide for a period of time sufficient to express the antibody in the host cells, and the antibody is optionally secreted into the culture medium in which the host cells are grown. After the antibody is secreted, standard protein purification methods can be used, for example, to recover and purify the antibody from the culture medium.

Drug and vaccine compositions:

The antibodies described herein can be administered as part of one or more pharmaceutical compositions. Unless any conventional carrier medium is incompatible with the antibodies of the invention, such as by producing any undesirable biological effect or interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is contemplated to be within the scope of the invention. Some examples of materials that can be used as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffer solutions, as well as other nontoxic compatible lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as colorants, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition (according to the judgment of the formulator).

The antibodies described herein are particularly suitable for the preparation of vaccine compositions. Therefore, another object of the present invention relates to a vaccine composition comprising the antibodies of the present invention.

In some embodiments, the vaccine composition of the present invention comprises an adjuvant. In some embodiments, the adjuvant is alum. In some embodiments, the adjuvant is incomplete Freund's adjuvant (IFA) or other oil-based adjuvants, and the weight ratio (w/w) thereof is 30-70%, preferably 40-60%, and more preferably 45-55%. In some embodiments, the vaccine composition of the present invention comprises at least one Toll-like receptor (TLR) agonist selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7 and TLR8 agonists.

Treatment:

The antibodies and pharmaceutical or vaccine compositions as described herein are particularly suitable for inducing an immune response against SARS-Cov-2 and can therefore be used for vaccine purposes.

Therefore, another object of the present invention relates to a method for vaccinating a subject in need thereof against SARS-Cov 2, comprising administering a therapeutically effective amount of an antibody of the present invention.

In some embodiments, the antibodies and drug or vaccine compositions described herein are particularly suitable for the treatment of Covid-19.

In some embodiments, the subject can be a human or any other animal (e.g., birds and mammals) susceptible to coronavirus infection (e.g., domestic animals such as cats and dogs; livestock and farm animals such as horses, cattle, pigs, chickens, etc.). Typically, the subject is a mammal, including non-primates (e.g., camels, donkeys, zebras, cattle, pigs, horses, goats, sheep, cats, dogs, rats and mice) and primates (e.g., monkeys, chimpanzees and humans). In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a farm animal or a pet. In some embodiments, the subject is a human. In some embodiments, the subject is a human infant. In some embodiments, the subject is a human child. In some embodiments, the subject is an adult. In some embodiments, the subject is an elderly person. In some embodiments, the subject is a premature infant.

In some embodiments, the subject can be symptomatic or asymptomatic.

Typically, the active ingredients of the invention (i.e., antibodies and pharmaceutical or vaccine compositions as described herein) are administered to a subject in a therapeutically effective amount. It should be understood that the total daily dosage of the compounds and compositions of the invention will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dosage level for any particular subject will depend on a variety of factors, including the condition to be treated and the severity of the condition; the activity of the specific compound employed; the specific composition employed, the age, weight, general health, sex and diet of the subject; the time of administration, route of administration and excretion rate of the specific compound employed; the duration of treatment; drugs used in combination or concurrently with the specific polypeptide employed; and similar factors well known in the medical field. For example, it is entirely within the technical scope of the art to start administering the compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the product can vary in the range of 0.01 to 1,000 mg per adult per day. In particular, the composition comprises 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of active ingredient for symptomatic adjustment of the dose of the subject to be treated. The drug generally comprises from about 0.01 mg to about 500 mg of active ingredient, particularly from 1 mg to about 100 mg of active ingredient. An effective amount of the drug is generally provided at a dosage level of from 0.0002 mg/kg to about 20 mg/kg body weight per day, particularly from about 0.001 mg/kg to 7 mg/kg body weight per day.

The antibodies and pharmaceutical or vaccine compositions described herein can be administered to a subject by any route of administration, particularly by oral, nasal, rectal, topical, buccal (e.g., sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal or intravenous) and transdermal administration, although the most appropriate route in any given case will depend on the nature and severity of the condition being treated, and the nature of the specific active agent being used.

In some embodiments, antibodies and drugs or vaccine compositions as described herein can be administered to a subject in combination with, for example, any known therapeutic agent or method for vaccination against SARS-Cov-2 coronavirus. Non-limiting examples of such known therapeutic agents include, but are not limited to, antiviral agents such as remdesivir, lopinavir, ritonavir, hydroxychloroquine, and chloroquine. In some embodiments, antibodies and drugs or vaccine compositions as described herein are administered in combination with immune checkpoint inhibitors. Examples of immune checkpoint inhibitors include PD-1 antagonists, PD-L1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, and BTLA antagonists. In some embodiments, PD-1 (Programmed Death-1) axis antagonists include PD-1 antagonists (e.g., anti-PD-1 antibodies), PD-L1 (Programmed Death Ligand-1) antagonists (e.g., anti-PD-L1 antibodies), and PD-L2 (Programmed Death Ligand-2) antagonists (e.g., anti-PD-L2 antibodies). In some embodiments, the anti-PD-1 antibody is selected from MDX-1106 (also known as Nivolumab, MDX-1106-04, ONO-4538, BMS-936558, and ), Merck 3475 (also known as Pembrolizumab, MK-3475, Lambrolizumab, and SCH-900475) and CT-011 (also known as Pidilizumab, hBAT, and hBAT-1). In some embodiments, the PD-1 binding antagonist is AMP-224 (also known as B7-DCIg). In some embodiments, the anti-PD-L1 antibody is selected from YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. MDX-1105 (also known as BMS-936559) is an anti-PD-L1 antibody described in WO 2007/005874. Antibody YW243.55.S70 is an anti-PD-L1 described in WO 2010/077634 A1. MEDI4736 is an anti-PD-L1 antibody described in WO2011/066389 and US2013/034559. MDX-1106 (also known as MDX-1106-04, ONO-4538 or BMS-936558) is an anti-PD-1 antibody described in U.S. Pat. No. 8,008,449 and WO2006/121168. Merck 3745 (also known as MK-3475 or SCH-900475) is an anti-PD-1 antibody described in U.S. Pat. No. 8,345,509 and WO2009/114335. CT-011 (pidilizumab) (also known as hBAT or hBAT-1) is an anti-PD-1 antibody described in WO2009/101611. AMP-224 (also known as B7-DCIg) is a PD-L2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342. Atezolizumab is an anti-PD-L1 antibody described in U.S. Pat. No. 8,217,149. Avelumab is an anti-PD-L1 antibody described in US20140341917. CA-170 is a PD-1 antagonist described in WO2015033301 & WO2015033299. Other anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,609,089, US2010028330 and/or US20120114649. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody selected from Nivolumab, Pembrolizumab or Pidilizumab. In some embodiments, the PD-L1 antagonist is selected from Avelumab, BMS-936559, CA-170, Durvalumab, MCLA-145, SP142, STI-A1011, STIA1012, STI-A1010, STI-A1014, A110, KY1003 and Atezolizumab, preferably Avelumab, Durvalumab or Atezolizumab.

The present invention will be further described by the following figures and examples. However, these examples and figures should not be interpreted as limiting the scope of the present invention in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Immunogenicity of aCD40-RBD vaccines given in homologous or heterologous primary/boost vaccination strategies. We purchased NSG humanized mice from Jackson Laboratories (USA). Five donors provided hematopoietic stem cells for human immune system reconstruction in mice. Animals were preserved at the Mondor Biomedical Research Institute (U955 INSERM-University of Paris-East Creteil, Île-de-France, France) according to the recommendations of the French Ministry of Higher Education, Research and Innovation. The local ethics committee ComethAnses/Enva/UPEC approved the protocol, license number 25329-2020051119073072v4.

Here, we tested whether such HIS mice could respond to CD40 targeting SARS-COV2 RBD protein to initiate B and T cell immunity when adjuvanted with TLR3/7 agonist (Poly(IC)). Poly(IC)/aCD.RBD was administered alone at weeks 0, 3, and 5 (Group 2), or Drep vaccine encoding SARS-CoV2S protein was used for primary immunization at week 0 and Poly(IC)/aCD.RBD was administered at weeks 3 and 5 (Group 3) (Figure 1A). Control group 1 consisted of half of the animals injected with PBS or Poly(IC). We monitored immune responses at day 21 (3 weeks after the primary injection) and at termination (one week after the last booster injection). Reconstitution of the human immune system was similar between the groups (Figure 1B: blood at baseline; Figure 1C: spleen at sacrifice).

Figure 2. Vaccine induces circulating antibody-secreting hu-B cells. The frequency of antibody-secreting cells (hCD45+hCD19+hCD27+hCD38+mCD45-) was evaluated by flow cytometry in the blood (A) of HIS mice and in the blood (B) and spleen (C) after sacrifice on day 21.

Figure 3. aCD40-RBD+Drep-S primary immunization elicits S-specific IgG+hu-B cells. The frequency of spike protein-specific IgG-converted hu-B cells (hCD45+hCD19+hIgG+spike protein+mCD45-) was evaluated in the blood of HIS mice (A) and in the spleen after sacrifice (B) on day 21 by flow cytometry using biotinylated SARS-CoV2 spike protein.

Figure 4. aCD40-RBD vaccine induced the expansion of CM CD4+hu-T cells at day 21 after immunization, and the appearance of EM CD4+T cells at day 42 after immunization. The frequencies of effector memory hu-CD4+T cells (hCD45+hCD3+hCD4+hCD27-hCD45RA-mCD45-) and central memory hu-CD4+T cells (hCD45+hCD3+hCD4+hCD27+hCD45RA-mCD45-) in the blood of HIS mice were evaluated by flow cytometry at baseline, day 21, and day of sacrifice.

Figure 5. The aCD40-RBD vaccine induced the expansion of CM CD4+hu-T cells at day 21 after immunization, and the appearance of EM CD4+T cells at day 42 after immunization. The frequencies of effector memory hu-CD8+T cells (hCD45+hCD3+hCD4-hCD27-hCD45RA-mCD45-) and central memory hu-CD8+T cells (hCD45+hCD3+hCD4+hCD27+hCD45RA-mCD45-) in the blood of HIS mice were evaluated by flow cytometry at baseline, day 21, and day of sacrifice.

Figure 6. The aCD40-RBD vaccine induced the expansion of CM CD4+hu-T cells at day 21 after immunization, and the appearance of EM CD4+T cells at day 42 after immunization. In HIS mice, the frequency of stem cell-like memory hu-CD8+T cells (hCD45+hCD3+hCD8+hTbet+hCD45RA+hCD62L+hCD95+hCD122+mCD45-) was evaluated by flow cytometry in the blood (A) and spleen (B) after sacrifice.

Figure 7. SARS-CoV-2-specific B and T cell responses induced by αCD40.RBD in convalescent NHPs. a. Study design in cynomolgus macaques. b. Relative MFI of IgG binding to SARS-CoV-2S protein in serum samples measured using a Luminex-based serological assay (mean ± SD of 6 animals per group). The vertical dashed lines represent virus exposure and vaccination, respectively. c Specific binding of SARS-CoV-2S protein in cynomolgus macaques (n = 12) before any exposure to SARS-CoV-2 (week -26) and at the week of vaccine injection (week 0) compared with convalescent humans (n = 7) sampled 24 weeks after symptom onset. The horizontal dashed line represents the background threshold and the bars represent the mean of each group. d. Quantification of SARS-CoV-2 antibodies against RBD measured in the serum of NHPs using a multiplex solid phase chemiluminescence assay. Each flat line represents an individual value and the thick dashed line represents the mean of each experimental group. e. Quantification of antibody-induced inhibition of ACE-2 binding in NHP serum. Symbols are the same as in d. f. The frequency of RBD-specific Th1 CD4+T cells (CD154+ and IFN-γ±IL-2±TNF-α) in the total CD4+T cell population for each non-immunized convalescent cynomolgus macaque (n=6, blue line and symbols) and αCD40.RBD-vaccinated convalescent cynomolgus macaque (n=6, green line and symbols). PBMCs were stimulated overnight with a library of overlapping SARS-CoV-2RBD peptides. The Wilcoxon signed-rank test was used to compare the time points of each experimental group. g. The frequency of cytokine-producing cells in RBD-specific CD4+T cells (CD154+) for non-immunized convalescent cynomolgus macaques (left) and αCD40.RBD-vaccinated convalescent cynomolgus macaques (right). Each bar represents the mean ± SD of 6 vaccinated convalescent cynomolgus macaques. The distribution of cytokines is shown within each bar. BL: baseline approximately 1 week before immunization; “Post imm.”: two weeks after immunization.

Figure 8. Efficacy of αCD40.RBD in convalescent cynomolgus monkeys.

a. Not infected Quantification of genomic viral RNA (gRNA) in tracheal swabs of (left, gray line), convalescent (middle, blue line), and convalescent cynomolgus macaques vaccinated with αCD40.RBD (right, green line). The bold line represents the mean viral load for each experimental group. b. Mean values of subgenomic (sgRNA) viral loads in tracheal swabs. Data are expressed as mean ± SD for each experimental group (n = 6 NHP/group). c. Percentage of cynomolgus macaques with viral gRNA exceeding the limit of detection (LOD) in tracheal swabs over time. The experimental groups were compared using the log-rank test; two-tailed p values are shown. d. Area under the curve (AUC) of gRNA viral loads in trachea (left panel) and nasopharyngeal swabs (right panel). e. Quantification of gRNA viruses in BAL three days after exposure (dpexpo). d, e Each graph represents one cynomolgus macaque (n = 6 NHP/group) and the bars represent the mean values for each group. Groups were compared using a two-tailed nonparametric Mann-Whitney test. f. Quantification of SARS-CoV-2 IgG binding to N, S and RBD after challenge. Each straight line represents an individual value, and the thick dashed line represents the mean of each experimental group. g. Quantification of antibody-induced inhibition of ACE-2 binding. Lines are as in f.

Embodiment 1:

Materials and methods

Female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) humanized mice (hu-mice) at 20 weeks of age were provided by Jackson Laboratories (Bar Harbor, ME, USA) as MTA#1720. Five donors provided hematopoietic stem cells for human immune system reconstitution in mice. The level of human immune cell reconstitution reached an average of 70%. Hu-mice were housed in microisolators at the Mondor Biomedical Research Infrastructure (U955 INSERM-Université Paris-Est Creteil, Île-de-France, France) and were artificially cared for under pathogen-free conditions, with a temperature of 20–24°C, 50%+/-15% humidity and a 12 h light/12 h dark cycle. These protocols were approved by the institutional ethics committee “Comité d’Ethique Anses/ENVA/UPEC (CEEA-016)” under statement number 20-043#25329. This research was authorized by the Department of Research, Innovation and Education, registration number 25329-2020051119073072v4.

Vaccination of humanized mice.

Hu- mice were immunized at 0, 3, and 5 weeks. The primary immunization injection was intraperitoneal administration of 10 μg of αCD40-RDB, with 50 μg of polyinosinic acid-polycytidylic acid (Poly-IC; Invivogen) as an adjuvant, with or without intramuscular injection of DREP-S (10 μg). Hu- mice then received booster intraperitoneal injections of αCD40-RDB (10 μg) and Poly-IC (50 μg). Blood was collected at week 0 (pre-immunization), week 3, and week 6. Hu- mice were euthanized at week 6.

Analysis of SARS-CoV-2 S protein-specific B cells

PBMCs from hu-mice three weeks after the primary immunization, and PBMCs and splenocytes from hu-mice at 6 weeks (one week after the last recall injection) were first incubated with biotinylated SARS-CoV-2 S protein for 30 minutes at 4° C. After a washing step, cells were stained with the following for 30 minutes at 4° C.: streptavidin-AF700 (1:10, Thermofisher Scientific), anti-human (h) CD45-PeCy7 (1:50, #2120080, HI30, Sony), anti-mouse (m) CD45-BV711 (1:50, #1115735, 30F11, Sony), anti-hCXCR4-Pe-Dazzle (1:50, #12-9999-42, 12G5, eBiosciences), anti-hCCR10-PE (1:50, #314305; R&D System), anti-CD3-BV510 (1:50, #2102240, UCHT1, Sony), anti-CD4-FITC (1:50, #2187040, OKT4, Sony), anti-CD8-PerCpCy5.5 (1:50, #2323550, SK1, Biolegend) antibodies and the following B cell specific antibodies: anti-hCD19-BV421 (1:16, #2111170, HIB19, Sony), anti-hCD20-APC (1:50, #2111550, 2H7, Sony), anti-hIgG-BV786 (1:16, #564230, G18-145, BD Biosciences), anti-hCD38-APC-Cy7 (1:16, #2117670, HIT2, Sony). Staining of splenocytes also included a viability marker (LiveDead aqueous or yellow stain, ThermoFisher Scientific). Cells were washed twice with FACS buffer (PBS 1% FCS) and acquired on a LSRII flow cytometer (BD Biosciences). Analysis was performed on FlowJo v.10.7.1.

result

The immunogenicity of aCD40-RBD vaccine (i.e., the antibody comprises i) a heavy chain fused to an RBD polypeptide and ii) a light chain as shown in SEQ ID NO: 43) administered in a homologous or heterologous primary/boost vaccination strategy was studied according to the protocol described in Figure 1. The results are shown in Figures 1 to 6. In particular, we show that the vaccine induces circulating antibody-secreting hu-B cells (Figure 2), induces S-specific IgG+hu-B cells (Figure 3), induces central memory CD4+hu-T cells to expand at day 21 after immunization and effector memory CD4+T cells to appear at day 42 after immunization (Figure 4), induces central memory CD8+hu-T cells to expand at day 21 after immunization and effector memory CD8+T cells to appear at day 42 after immunization (Figure 5), and finally induces stem cell-like memory hu-CD8+T cells at day 42 after immunization (Figure 6). In fact, a single intraperitoneal injection of αCD40.RBD (10 μg) with polyinosinic acid-polycytidylic acid (Poly-IC; 50 μg) was sufficient to induce SARS-CoV-2S protein-specific IgG conversion of human B cells in the blood of 50% of the immunized mice (Figure 3). At week 6 (i.e., one week after the last αCD40.RBD boost), unbiased t-SNE analysis of human CD19+B cells in the spleen showed that the cell clusters in the vaccine group corresponded to the well-described terminally differentiated plasma cells (PC), early plasmablasts (PB), and a subset of PB and immature PC, but not in the control group (data not shown). At the same time point, SARS-CoV-2S protein-specific IgG conversion of human B cells in the spleen was detected in all vaccinated hu-mice (data not shown), mainly PB and immature PC phenotypes.

All spike protein-specific IgG-converted human B cells expressed CXCR4, and discrete cell islands were observed in t-SNE analysis driven by high expression of CCR10 (data not shown), which was confirmed by manual reverse gating (data not shown). We next evaluated the ability of the vaccine to induce specific and functional CD4+ and CD8+ memory T cells. After in vitro stimulation of splenocytes with the RBD peptide library, Th1 (IFN-γ+/-IL-2+/-TNF-α) type CD4+T-cell responses and IFNγ-secreting CD8+T cells were observed for vaccinated hu-mice (data not shown). We used HLA-I tetramers to confirm the presence of human CD8+T cells specific for the optimal epitope predicted for the SARS-CoV-2RBD protein in the spleens of vaccinated hu-mice (data not shown). Subunit vaccines can also be considered as boosters for other types of vaccines in human vaccination campaigns. Therefore, in addition to homologous prime-boost regimens, we also tested the ability of αCD40.RBD to boost heterologous primary immunization using a vector-based vaccine. The DNA-launched self-amplifying RNA replicon vector encoding the SARS-CoV-2 spike glycoprotein (DREP)-S is a previously described ^platform28 that is based on the alphavirus genome with genes encoding the viral RNA replicase but lacking genes encoding the structural proteins of the ^virus29 .

We demonstrated that the prime-boost strategy containing αCD40.RBD effectively elicited SARS-COV-2-specific responses in both B- and T-cells in both vaccinated groups (Figure 2). In both vaccinated groups, we showed expansion of effector memory CD4 and CD8+ T cells (CD45RA ^{- CD27-} ).

Example 2: In convalescent cynomolgus monkeys, the αCD40.RBD vaccine again elicited a specific immune response and improved Protection from SARS-CoV-2 reinfection in convalescent cynomolgus macaques

Materials and methods

This study used cynomolgus macaques ( Macaca fascicularis ) (8 females and 13 males) aged 37-58 months, from an AAALAC-accredited breeding center in Mauritius. All animals were housed in the IDMIT facility (CEA, Fontenay-aux-roses) in BSL-3 containers (Animal Facility Authorization # D92-032-02, Seine, France) in compliance with European Directive 2010/63/EU, French regulations of the Office for Laboratory Animal Welfare and Standards for Human Care and Use of Laboratory Animals, of the Office for Laboratory Animal Welfare (OLAW, Assurance Number # A5826-01, USA). The protocols were approved by the institutional ethical committee “Comité d’Ethique en Expérimentation Animaledu Commissariat à l’Energie Atomique et aux Energies Alternatives” (CEtEA#44) under statement number A20-011. The study was authorized by the “Ministry of Research, Innovation and Education” under registration number APAFIS#24434-2020030216532863v1.

Nonhuman primate study design.

Convalescent cynomolgus monkeys previously exposed to SARS-COV-2 were used to evaluate the antiviral efficacy of hydroxychloroquine (HCQ) and azithromycin (AZTH). Neither AZTH, HCQ, nor the combination of HCQ and AZTH showed a significant effect on viral replication5. Six months post-infection (p.i.) (24-26 weeks), 12 of these animals were randomly assigned to two experimental groups. The convalescent vaccinated group (n=6) received 200μg of αCD40.RBD vaccine diluted in PBS without any adjuvant by the subcutaneous (SC) route. The other six convalescent animals served as controls and received an equal amount of PBS by SC. At weeks 2 and 4 after vaccine or PBS injection, samples were taken from both groups of convalescent animals to evaluate anti-SARS-CoV-2 immune responses. An additional 6 age-matched (43.7±6.76 months) cynomolgus monkeys from the same source were included in the study as controls that were not infected with any exposure to SARS-CoV-2.

Experimental infection of cynomolgus macaques with SARS-CoV-2.

Four weeks after immunization, all animals were exposed to a total dose of 106 pfu of SARS-CoV-2 virus (hCoV-19/France/lDF0372/2020 strain; GISAID EpiCoV platform, accession number EPI_ISL_406596) via a combined intranasal and intratracheal route (0.25 mL per nostril, 4.5 mL intratracheally, i.e., 5 mL in total; day 0), pretreated with atropine (0.04 mg/kg), anesthetized with ketamine (5 mg/kg) and medetomidine (0.05 mg/kg). Nasopharyngeal, tracheal, and rectal swabs were collected on days 1, 2, 3, 4, 6, 9, 14, and 20 post-exposure (dpexp.), while blood was collected on days 2, 4, 6, 9, 14, and 20 post-exposure. On day 3 post-exposure, bronchoalveolar lavage (BAL) was performed using 50 mL sterile saline to approach the peak of viral replication and to be able to observe differences between the vaccinated and control groups. In our earlier studies30, we found that viral loads in BAL were very low or negative at later time points. Chest CT was performed at baseline and on days 2 and 6 post-exposure, and animals were anesthetized with tiletamine (4 mg/kg) and zolazepam (4 mg/kg). Lesions were scored as we previously ^described30 . Blood cell counts, hemoglobin, and hematocrit were determined from EDTA blood using a DXH800 analyzer (Beckman Coulter).

Evaluation of anti-Spike, anti-RBD, and IgG inhibitory antibodies.

Anti-spike protein IgG from human and NHP sera was titrated by multiple bead assay. In brief, Luminex beads were coupled to the spike protein and added to the Bio-Plex plate (BioRad) as described previously6. The beads were washed with PBS 0.05% Tween using a magnetic plate washer (MAG2x program) and incubated with serially diluted individual sera for 1 hour. The beads were then washed and anti-NHP IgG-PE secondary antibody (Southern Biotech, clone SB108a) was added at a 1:500 dilution at room temperature for 45 minutes. After washing, the beads were resuspended in read buffer for 5 minutes on a plate shaker with stirring (800 rpm) and then read directly on a Luminex Bioplex200 microplate reader (Biorad). The average MFI from the baseline sample was used as a reference value for the negative control. The amount of IgG against the spike protein is reported as the MFI signal divided by the average signal of the negative control. Human serum was collected from hospitalized patients with virologically confirmed COVID-19 3 months after symptom recovery and used as a control for titrating anti-spike protein antibodies.

Anti-RBD and anti-nucleocapside (N) IgG were titrated using a commercially available multiplex immunoassay developed by Messoscale Discovery (MSD, Rockville, MD) as previously described7. Briefly, 200-400 μg/mL of antigen in a proprietary buffer was spotted, washed, dried, and packaged for further use ( Coronavirus Plate 2). The plate was then blocked with MSD Blocker A, followed by the addition of reference standards, controls, and samples diluted 1:500 and 1:5000 in dilution buffer. After incubation, detection antibody (MSD SULFO-TAGTM anti-human IgG antibody) was added, followed by MSD GOLDTM Reading Buffer B, and the plate was read using a MESO QuickPlex SQ 120MM reader. Results are expressed as arbitrary units (AU)/mL.

The MSD pseudoneutralization assay is used to measure antibodies that neutralize the binding of the spike protein to the ACE2 receptor. The plates are blocked and washed as described above, and the assay calibrants (COVID-19 neutralizing antibodies; monoclonal antibodies against the S protein; 200 μg/mL), control serum, and test serum samples diluted 1:10 and 1:100 in assay diluent are added to the plates. After the plate incubation, 0.25 μg/ml of MSD SULFO-TAGTM solution coupled to ACE-2 is added, after which the plates are read as described above. The electrochemiluminescence (ECL) signal is recorded and the results are expressed as 1/ECL.

Statistical analysis

Data were collected using classic Excel files (Microsoft Excel 2016). Differences between unmatched groups were compared using unpaired t-tests or Mann-Whitney U tests (Graphpad Prism 8.0), and differences between matched groups were compared using paired t-tests or Wilcoxon signed-rank tests (Graphpad Prism 8.0). Viral kinetic parameters were compared using the log-rank test (Graphpad Prism 8.0). Correlations between viral and immune parameters were determined using nonparametric Spearman correlations (GraphpadPrism 8.0).

result

The immunogenicity observed in the hu-mouse model is consistent with that of our previous CD40-targeted influenza and HIV vaccine ^{studies21,22,26,27} , demonstrating that αCD40.RBD can be an effective primary or boost vaccine to elicit RBD-specific T and B cell ^responses19 . Therefore, we injected 200 μg of the unadjuvanted vaccine subcutaneously into six convalescent cynomolgus macaques. An additional 12 animals (6 convalescent and 6 uninfected) were injected with PBS as a control (Figure 7a). All convalescent cynomolgus macaques, randomized between vaccine and control groups, had been infected with SARS-CoV-2 approximately six months previously (range = 26–24 weeks) in a study evaluating the use of hydroxychloroquine (HCQ) for pre- or post-exposure prophylaxis. No evidence of antiviral efficacy of HCQ was observed30 ^, and all animals developed similar viral loads after the first exposure to the virus (data not shown) and had a transient and moderate disease, resulting in elevated levels of anti-S IgG antibodies detected in the serum (Figure 7b). At the time of αCD40.RBD vaccination, anti-S IgG levels in the two groups of convalescent cynomolgus macaques were comparable, with specific responses detected in the average range of convalescent patient sera (Figure 7c).

Before vaccination, SARS-CoV-2 infected cynomolgus monkeys produced anti-RBD antibodies (Figure 7d), as well as low but detectable antibodies that inhibit the binding of the spike protein to the ACE2 receptor (Figure 7e). Before vaccination, low Th1 (IFN-γ+/-IL-2+/-TNF-α) type CD4+T cell responses were observed in both groups of recovering cynomolgus monkeys after ex vivo stimulation of PBMCs with RBD and N peptide libraries (data not shown). No anti-RBD or anti-N CD8+T cells were detected in the recovering animals (data not shown). Two weeks after injection of the αCD40.RBD vaccine, the levels of anti-S (Figure 7b) and anti-RBD IgG (Figure 7d) in the serum of all six vaccinated cynomolgus monkeys increased significantly, which was associated with an increased ability to inhibit the binding of RBD to the ACE2 receptor (p=0.022, Figure 7e), as it remained elevated four weeks after vaccination. In an in vitro assay using real virus ¹⁴ , we demonstrated that vaccine-generated antibodies not only neutralized variants with D614G present in αCD40.RBD (data not shown), but also cross-neutralized B1.1.7 and, to a lesser extent, B1.351 (known to be partially resistant to antibodies generated by previously circulating variants ^31,32 ). None of these parameters increased in the convalescent controls injected with PBS (Fig. 7b, Fig. 7d, Fig. 7e). In addition, anti-S IgG levels in vaccinated cynomolgus macaques (p=0.0018) were higher than those typically observed in humans 1 to 3 months after symptomatic SARS-CoV-2 infection (data not shown). Immunization also induced a significant increase in anti-RBD Th1 responses in all six immunized animals (P = 0.031; Figure 7f, Figure 7g), while there was no change in the levels of anti-N CD4+ T cells (data not shown) or SARS-CoV-2-specific CD8+ T cells (data not shown).

Four weeks after vaccine or placebo injection, 12 convalescent cynomolgus macaques were exposed a second time to a high dose (1 × 106 pfu) of SARS-CoV-2, administered via a combined intranasal and intratracheal route using a previously reported challenge procedure ^30. Six SARS-COV-2-naive animals were also challenged as controls. All uninfected animals became infected, as shown by the detection of viral genomic (gRNA) and subgenomic (sgRNA) RNA in tracheal (Figure 8a–8d) and nasopharyngeal (Figure 8d) swabs and bronchoalveolar lavage (BAL, Figure 8e). Notably, the dynamics of viral replication in these animals were comparable to those observed in the first infection of both groups of convalescent cynomolgus macaques 6 months earlier (data not shown).

Unvaccinated convalescent animals were not protected against a second SARS-CoV-2 attack, but significantly lower levels of viral RNA were detected in the upper respiratory tract than in uninfected animals (Fig. 8a-Fig. 8e). The αCD40.RBD vaccine significantly improved partial protection in convalescent cynomolgus macaques. All vaccinated animals showed significantly lower levels of viral gRNA compared to unvaccinated convalescent animals (P = 0.015, Fig. 8d). For 5 of the 6 vaccinated animals, sgRNA levels in upper respiratory tract samples remained below the limit of detection, while sgRNA was detected in 4 of the 6 unvaccinated convalescent animals and in all uninfected control animals (data not shown). In addition, the time post-exposure (p.expo.) required to reach undetectable gRNA levels was significantly shorter in vaccinated convalescent animals compared to unvaccinated control animals (Fig. 8c). Vaccination efficacy was also high in the lower respiratory tract, as only 3 of 6 vaccinated cynomolgus macaques had gRNA above the limit of detection in the BAL on day 3 postexposure, whereas all 6 unvaccinated convalescent animals had gRNA above the limit of detection on day 6 (Fig. 8e). Complete protection against viral shedding from the gastrointestinal tract was seen in both unvaccinated and vaccinated convalescent cynomolgus macaques (data not shown), suggesting that immunity from natural infection, in addition to vaccines, can play an important role in preventing secondary viral ^{transmission33} .

In vaccinated and non-vaccinated convalescent cynomolgus monkeys, the reduction in viral load was associated with limited effects on leukocyte counts (data not shown) and reduced plasma cytokine concentrations, particularly those of IL-1RA and CCL2 (data not shown), compared with uninfected animals. This viral load and cytokine profile was also associated with a reduction in lung lesions (data not shown), as scored by X-ray computed tomography (CT).

We then analyzed the immune response of all animals after SARS-CoV-2 virus challenge. Uninfected controls showed the slowest progression of anti-S, anti-RBD, and anti-N IgG (Figure 8f), and their levels remained significantly lower than those of the other two groups at day 20 after exposure (p = 0.022). Convalescent animals that were not vaccinated developed a rapid and robust memory antibody response (Figure 8f), which was associated with a significant increase in the ability of serum to neutralize ACE2 binding to RBD at day 9 after exposure (p = 0.031), reaching the levels observed in the vaccinated group at day 9 after exposure. In vaccinated cynomolgus macaques, serum anti-S and anti-RBD-specific antibody responses and neutralizing activity remained at high levels already achieved at the time of challenge and remained superior to those of control cynomolgus macaques (Figure 8f, Figure 8g). For most control (recovering and uninfected) animals, anti-RBD Th1 CD4+ responses increased after challenge, with some uninfected controls having high levels as early as day 9 after exposure (data not shown). In contrast, all 18 animals showed comparable antibody and CD4+ T cell responses to the N peptide pool (data not shown), which may reflect the predominance of responses to nonstructural antigens in infected individuals. IFN-γ-mediated CD8+ T cell responses were also primarily directed against the N peptide (data not shown), but were significantly less intense in all recovered cynomolgus macaques than in uninfected controls (data not shown), likely reflecting reduced exposure to viral antigens due to better control of viral replication. Spearman analysis between all recorded parameters showed that the induction of anti-RBD and ACE2 inhibitory antibodies was the strongest parameter associated with reduced viral load and disease markers, as were plasma levels of the inflammatory cytokines IL-1RA and CCL2 (data not shown).

discuss

In humans, the durability of protection elicited by natural SARS-CoV-2 infection and by the first vaccine candidates is unknown. In convalescent humans, waning virus-neutralizing antibody responses have been reported months after previous exposure and upon ^{reinfection33,34} . A decline in neutralizing antibody levels has been observed in most patients within three months of infection, which may indicate the need for booster vaccines to provide durable ^protection35 . In contrast to ^previous NHP rechallenge studies conducted shortly after initial infection36 , we demonstrated that convalescent cynomolgus macaques were not completely protected against SARS-CoV-2 reinfection six months after initial exposure, confirming that protective immunity wanes over time. Furthermore, current vaccines for use in humans are designed to prevent severe disease, and only partial information is available on their ability to prevent infection and reduce initial viral replication to levels required to significantly limit secondary transmission. Vaccinated individuals who develop asymptomatic or mildly symptomatic infection can continue to shed the virus and actively contribute to viral circulation. The αCD40.RBD vaccine we developed significantly boosted immunity in convalescent cynomolgus macaques, allowing viral loads to drop to levels that would prevent such secondary transmission upon reinfection. Such vaccines may therefore represent an appropriate boost to pre-existing immunity, either induced by natural infection or by previous primary immunization with vector-based vaccines. This new generation of subunit vaccines, targeting antigens on cells expressing CD40, may have the advantage of being a safe and highly effective boosting strategy. The ability to induce protective immunity without the need for an adjuvant will accelerate the development of protein-based vaccines, which are expected to be more well tolerated than adjuvanted vaccines and are therefore suitable for use in people with specific susceptibilities and children, who are important populations to consider in controlling viral circulation.

References:

Throughout the application, various references describe the state of the art to which the present invention relates. The disclosures of these references are incorporated into the present disclosure by reference.

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Sequence Listing

<110> National Institute of Health Sciences

<120> Antibodies coupled or fused to the receptor binding domain of the SARS-COV-2 spike protein and their use for vaccine purposes

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<170> PatentIn version 3.3

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Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

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Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

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Val Leu Lys Gly Val Lys Leu His Tyr Thr

1265 1270

<210> 2

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> H-CDR1

<400> 2

Gly Phe Thr Phe Ser Asp Tyr Tyr Met Tyr

1 5 10

<210> 3

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> H-CDR2

<400> 3

Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val Lys

1 5 10 15

Gly

<210> 4

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> H-CDR3

<400> 4

Arg Gly Leu Pro Phe His Ala Met Asp Tyr

1 5 10

<210> 5

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> L-CDR1

<400> 5

Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 6

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> L-CDR2

<400> 6

Tyr Thr Ser Ile Leu His Ser

1 5

<210> 7

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> L-CDR3

<400> 7

Gln Gln Phe Asn Lys Leu Pro Pro Thr

1 5

<210> 8

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> H-CDR1

<400> 8

Gly Tyr Ser Phe Thr Gly Tyr Tyr Met His

1 5 10

<210> 9

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> H-CDR2

<400> 9

Arg Ile Asn Pro Tyr Asn Gly Ala Thr Ser Tyr Asn Gln Asn Phe Lys

1 5 10 15

Asp

<210> 10

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> H-CDR3

<400> 10

Glu Asp Tyr Val Tyr

1 5

<210> 11

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> L-CDR1

<400> 11

Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His

1 5 10 15

<210> 12

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> L-CDR2

<400> 12

Lys Val Ser Asn Arg Phe

1 5

<210> 13

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> L-CDR3

<400> 13

Ser Gln Ser Thr His Val Pro Trp Thr

1 5

<210> 14

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> H-CDR1

<400> 14

Gly Tyr Thr Phe Thr Asp Tyr Val Leu His

1 5 10

<210> 15

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> H-CDR2

<400> 15

Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 16

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> H-CDR3

<400> 16

Gly Tyr Pro Ala Tyr Ser Gly Tyr Ala Met Asp Tyr

1 5 10

<210> 17

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> L-CDR1

<400> 17

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 18

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> L-CDR2

<400> 18

Tyr Thr Ser Arg Leu His Ser

1 5

<210> 19

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> L-CDR3

<400> 19

His His Gly Asn Thr Leu Pro Trp Thr

1 5

<210> 20

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> VH domain

<400> 20

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Lys Gln Ala His Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asn Pro Tyr Asn Gly Ala Thr Ser Tyr Asn Gln Asn Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Tyr Val Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

Ser Ser Ala Ser

115

<210> 21

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> VL domain

<400> 21

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys

100 105

<210> 22

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> VH domain

<400> 22

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asn Pro Tyr Asn Gly Ala Thr Ser Tyr Asn Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Tyr Val Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

Ser Ser Ala Ser

115

<210> 23

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<223> VH domain

<400> 23

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Val Leu His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Tyr Pro Ala Tyr Ser Gly Tyr Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser

115 120

<210> 24

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> VL domain

<400> 24

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys His His Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys

100

<210> 25

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> VH domain

<400> 25

Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Phe Tyr Tyr Gly Tyr Ser Phe Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Ala Ser

115 120

<210> 26

<211> 102

<212> PRT

<213> Artificial sequence

<220>

<223> VL domain

<400> 26

Gln Ile Val Leu Thr Gln Ser Pro Ala Phe Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys

100

<210> 27

<211> 128

<212> PRT

<213> Artificial sequence

<220>

<223> VH domain

<400> 27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asp Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Asn Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gln Pro Leu Gly Tyr Cys Thr Asn Gly Val Cys Ser Tyr

100 105 110

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

115 120 125

<210> 28

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> VL domain

<400> 28

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Tyr Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Thr Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ile Phe Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys

100

<210> 29

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> VH domain

<400> 29

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Leu Pro Phe His Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser

115 120

<210> 30

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> VL domain

<400> 30

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Lys Leu Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys

100

<210> 31

<211> 81

<212> PRT

<213> Artificial sequence

<220>

<223> VH domain

<400> 31

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

1 5 10 15

Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

20 25 30

Gly Asp Ile Tyr Pro Gly Ser Gly Tyr Ser Phe Tyr Asn Glu Asn Phe

35 40 45

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr

50 55 60

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

65 70 75 80

Ala

<210> 32

<211> 76

<212> PRT

<213> Artificial sequence

<220>

<223> VL domain

<400> 32

Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly

1 5 10 15

Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys

20 25 30

Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg

35 40 45

Phe Ser Gly Ser Gly Ser Gly Thr Asn Phe Thr Leu Lys Ile Ser Arg

50 55 60

Val Glu Ala Glu Asp Leu Gly Leu Tyr Phe Cys Ser

65 70 75

<210> 33

<211> 82

<212> PRT

<213> Artificial sequence

<220>

<223> VH domain

<400> 33

Ser Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp

1 5 10 15

Tyr Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp

20 25 30

Ile Gly Asp Ile Tyr Pro Gly Ser Gly Tyr Ser Phe Tyr Asn Glu Asn

35 40 45

Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala

50 55 60

Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe

65 70 75 80

Cys Ala

<210> 34

<211> 74

<212> PRT

<213> Artificial sequence

<220>

<223> VL domain

<400> 34

Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn

1 5 10 15

Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly Leu

20 25 30

Ile Gly Gly Thr Asn Asn Arg Val Ser Gly Val Pro Ala Arg Phe Ser

35 40 45

Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln

50 55 60

Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala

65 70

<210> 35

<211> 456

<212> PRT

<213> Artificial sequence

<220>

<223> Heavy chain

<400> 35

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Asp His

20 25 30

Asp Met His Trp Val Gln Gln Thr Pro Val Tyr Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Asp Pro Glu Thr Gly Asp Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Ile Leu Thr Ala Asp Lys Ser Ser Arg Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Ile Pro Phe Tyr Tyr Ser Asn Tyr Ser Pro Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Ala Leu Val Thr Val Ser Ala Ala Lys Thr Thr Ala Pro Ser

115 120 125

Val Tyr Pro Leu Ala Pro Val Cys Gly Gly Thr Thr Gly Ser Ser Val

130 135 140

Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu

145 150 155 160

Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala

165 170 175

Leu Leu Gln Ser Gly Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr

180 185 190

Ser Asn Thr Trp Pro Ser Gln Thr Ile Thr Cys Asn Val Ala His Pro

195 200 205

Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Val Pro Ile

210 215 220

Thr Gln Asn Pro Cys Pro Pro Leu Lys Glu Cys Pro Pro Cys Ala Asp

225 230 235 240

Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp

245 250 255

Val Leu Met Ile Ser Leu Ser Pro Met Val Thr Cys Val Val Val Asp

260 265 270

Val Ser Glu Asp Asp Pro Asp Ala Gln Ile Ser Trp Phe Val Asn Asn

275 280 285

Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn

290 295 300

Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp

305 310 315 320

Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Arg Ala Leu Pro

325 330 335

Ser Pro Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Pro Val Arg Ala

340 345 350

Pro Gln Val Tyr Val Leu Pro Pro Pro Ala Glu Glu Met Thr Lys Lys

355 360 365

Glu Phe Ser Leu Thr Cys Met Ile Thr Gly Phe Leu Pro Ala Glu Ile

370 375 380

Ala Val Asp Trp Thr Ser Asn Gly Arg Thr Glu Gln Asn Tyr Lys Asn

385 390 395 400

Thr Ala Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys

405 410 415

Leu Arg Val Gln Lys Ser Thr Trp Glu Arg Gly Ser Leu Phe Ala Cys

420 425 430

Ser Val Val His Glu Gly Leu His Asn His Leu Thr Thr Lys Thr Ile

435 440 445

Ser Arg Ser Leu Gly Lys Ala Ser

450 455

<210> 36

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> Light chain

<400> 36

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Arg Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala Pro

100 105 110

Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly

115 120 125

Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn

130 135 140

Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn

145 150 155 160

Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser

165 170 175

Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr

180 185 190

Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe

195 200 205

Asn Arg Asn Glu Cys

210

<210> 37

<211> 27

<212> PRT

<213> Artificial sequence

<220>

<223> Connector

<400> 37

Gln Thr Pro Thr Asn Thr Ile Ser Val Thr Pro Thr Asn Asn Ser Thr

1 5 10 15

Pro Thr Asn Asn Ser Asn Pro Lys Pro Asn Pro

20 25

<210> 38

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Connector

<400> 38

Ser Ser Val Ser Pro Thr Thr Ser Val His Pro Thr Pro Thr Ser Val

1 5 10 15

Pro Pro Thr Pro Thr Lys Ser Ser Pro

20 25

<210> 39

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Connector

<400> 39

Pro Thr Ser Thr Pro Ala Asp Ser Ser Thr Ile Thr Pro Thr Ala Thr

1 5 10 15

Pro Thr Ala Thr Pro Thr Ile Lys Gly

20 25

<210> 40

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Connector

<400> 40

Thr Val Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ile Val Thr Thr

1 5 10 15

Ile Thr Pro Thr Ala Thr Thr Lys Pro

20 25

<210> 41

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Connector

<400> 41

Thr Asn Gly Ser Ile Thr Val Ala Ala Thr Ala Pro Thr Val Thr Pro

1 5 10 15

Thr Val Asn Ala Thr Pro Ser Ala Ala

20 25

<210> 42

<211> 671

<212> PRT

<213> Artificial sequence

<220>

<223> h anti-CD40VH3-LV-hIgG4H-C-AS-virus SARS-CoV-2-spike protein-RBD-EPEA

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Leu Pro Phe His Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser

435 440 445

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

450 455 460

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

465 470 475 480

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

485 490 495

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

500 505 510

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

515 520 525

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

530 535 540

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

545 550 555 560

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

565 570 575

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

580 585 590

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

595 600 605

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

610 615 620

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

625 630 635 640

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

645 650 655

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Ser Val Asn Phe

660 665 670

<210> 43

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> h anti-CD40VK2-LV-hIgGK-C

<400> 43

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Lys Leu Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 44

<211> 439

<212> PRT

<213> Artificial sequence

<220>

<223> h anti-CD40VK2-LV-hIgGK-C-virus SARS-CoV-2-spike protein-RBDC221S SA variant

<400> 44

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Lys Leu Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Ala Ser Arg Val Gln Pro Thr Glu Ser Ile

210 215 220

Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe

225 230 235 240

Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile

245 250 255

Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe

260 265 270

Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu

275 280 285

Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu

290 295 300

Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn

305 310 315 320

Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser

325 330 335

Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg

340 345 350

Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr

355 360 365

Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe

370 375 380

Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly

385 390 395 400

Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu

405 410 415

His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val

420 425 430

Lys Asn Lys Ser Val Asn Phe

435

<210> 45

<211> 671

<212> PRT

<213> Artificial sequence

<220>

<223> h anti-CD40VH3-LV-hIgGK-C-virus SARS-CoV-2-spike protein-RBDC221S SA variant

<400> 45

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Leu Pro Phe His Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser

435 440 445

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

450 455 460

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

465 470 475 480

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

485 490 495

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

500 505 510

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

515 520 525

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

530 535 540

Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

545 550 555 560

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

565 570 575

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

580 585 590

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

595 600 605

Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

610 615 620

Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val

625 630 635 640

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

645 650 655

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Ser Val Asn Phe

660 665 670

<210> 46

<211> 439

<212> PRT

<213> Artificial sequence

<220>

<223> h anti-CD40VK2-LV-hIgG4H-C-virus SARS-CoV-2-spike protein-RBDC221S

<400> 46

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Lys Leu Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Ala Ser Arg Val Gln Pro Thr Glu Ser Ile

210 215 220

Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe

225 230 235 240

Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile

245 250 255

Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe

260 265 270

Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu

275 280 285

Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu

290 295 300

Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn

305 310 315 320

Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser

325 330 335

Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg

340 345 350

Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr

355 360 365

Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe

370 375 380

Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly

385 390 395 400

Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu

405 410 415

His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val

420 425 430

Lys Asn Lys Ser Val Asn Phe

435

<210> 47

<211> 149

<212> PRT

<213> Artificial sequence

<220>

<223> CD40L binding domain

<400> 47

Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser

1 5 10 15

Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly

20 25 30

Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln

35 40 45

Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr

50 55 60

Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser

65 70 75 80

Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala

85 90 95

Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His

100 105 110

Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn

115 120 125

Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe

130 135 140

Gly Leu Leu Lys Leu

145

<210> 48

<211> 439

<212> PRT

<213> Artificial sequence

<220>

<223> h anti-CD40VK2-LV-hIgGK-C-virus SARS-CoV-2-spike protein-RBDC221S

(K417N, L452R, T478K, E484Q, N501Y)

<400> 48

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Lys Leu Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Ala Ser Arg Val Gln Pro Thr Glu Ser Ile

210 215 220

Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe

225 230 235 240

Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile

245 250 255

Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe

260 265 270

Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu

275 280 285

Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu

290 295 300

Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn

305 310 315 320

Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser

325 330 335

Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Arg Tyr Arg

340 345 350

Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr

355 360 365

Glu Ile Tyr Gln Ala Gly Ser Lys Pro Cys Asn Gly Val Gln Gly Phe

370 375 380

Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly

385 390 395 400

Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu

405 410 415

His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val

420 425 430

Lys Asn Lys Ser Val Asn Phe

435

<210> 49

<211> 671

<212> PRT

<213> Artificial sequence

<220>

<223> h anti-CD40VH3-LV-hIgGK-C-virus SARS-CoV-2-spike protein-RBDC221S

(K417N, L452R, T478K, E484Q, N501Y)

<400> 49

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Leu Pro Phe His Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser

435 440 445

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

450 455 460

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

465 470 475 480

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

485 490 495

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

500 505 510

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

515 520 525

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

530 535 540

Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

545 550 555 560

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

565 570 575

Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

580 585 590

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys

595 600 605

Pro Cys Asn Gly Val Gln Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

610 615 620

Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val

625 630 635 640

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

645 650 655

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Ser Val Asn Phe

660 665 670

<210> 50

<211> 439

<212> PRT

<213> Artificial sequence

<220>

<223> h anti-CD40VK2-LV-hIgGK-C-virus SARS-CoV-2-spike protein-RBDC221S

(K417N, L452R, T478K, E484K, N501Y)

<400> 50

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Lys Leu Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Ala Ser Arg Val Gln Pro Thr Glu Ser Ile

210 215 220

Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe

225 230 235 240

Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile

245 250 255

Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe

260 265 270

Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu

275 280 285

Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu

290 295 300

Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn

305 310 315 320

Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser

325 330 335

Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Arg Tyr Arg

340 345 350

Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr

355 360 365

Glu Ile Tyr Gln Ala Gly Ser Lys Pro Cys Asn Gly Val Lys Gly Phe

370 375 380

Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly

385 390 395 400

Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu

405 410 415

His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val

420 425 430

Lys Asn Lys Ser Val Asn Phe

435

<210> 51

<211> 671

<212> PRT

<213> Artificial sequence

<220>

<223> h anti-CD40VH3-LV-hIgGK-C-virus SARS-CoV-2-spike protein-RBDC221S

(K417N, L452R, T478K, E484K, N501Y)

<400> 51

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Leu Pro Phe His Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser

435 440 445

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

450 455 460

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

465 470 475 480

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

485 490 495

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

500 505 510

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

515 520 525

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

530 535 540

Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

545 550 555 560

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

565 570 575

Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

580 585 590

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys

595 600 605

Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

610 615 620

Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val

625 630 635 640

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

645 650 655

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Ser Val Asn Phe

660 665 670

<210> 52

<211> 439

<212> PRT

<213> Artificial sequence

<220>

<223> h anti-CD40VK2-LV-hIgGK-C-virus SARS-CoV-2-spike protein-RBDC221S (K417N,

E484Q, N501Y)

<400> 52

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Lys Leu Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys Ala Ser Arg Val Gln Pro Thr Glu Ser Ile

210 215 220

Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe

225 230 235 240

Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile

245 250 255

Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe

260 265 270

Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu

275 280 285

Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu

290 295 300

Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn

305 310 315 320

Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser

325 330 335

Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg

340 345 350

Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr

355 360 365

Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Gln Gly Phe

370 375 380

Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly

385 390 395 400

Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu

405 410 415

His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val

420 425 430

Lys Asn Lys Ser Val Asn Phe

435

<210> 53

<211> 671

<212> PRT

<213> Artificial sequence

<220>

<223> h anti-CD40VH3-LV-hIgGK-C-virus SARS-CoV-2-spike protein-RBDC221S (K417N,

E484Q, N501Y)

<400> 53

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Leu Pro Phe His Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser

435 440 445

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

450 455 460

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

465 470 475 480

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

485 490 495

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

500 505 510

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

515 520 525

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

530 535 540

Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

545 550 555 560

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

565 570 575

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

580 585 590

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

595 600 605

Pro Cys Asn Gly Val Gln Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

610 615 620

Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val

625 630 635 640

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

645 650 655

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Ser Val Asn Phe

660 665 670

Claims (43)

1. An antibody directed against the surface antigen of an antigen-presenting cell, wherein the heavy chain and/or light chain is coupled or fused to an RBD polypeptide with the amino acid residue from position 319 in SEQ ID NO: 1 Amino acid sequences ranging from amino acid residues to position 541 have at least 90% identity. 2. The antibody of claim 1, wherein the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1. 3. The antibody of claim 1, wherein the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1 and comprising C538S non- Naturally occurring mutations. 4. The antibody of claim 1, wherein the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1 and comprising C538S non- Naturally occurring mutations and one or more naturally occurring mutations selected from the group consisting of K417N, K417T, E484K and N501Y mutations. 5. The antibody of claim 1, wherein the RBD polypeptide is composed of amino acids ranging from the amino acid residue at position 319 to the amino acid residue at position 541 in SEQ ID NO: 1, and includes K417T, E484K, N501Y naturally occurring mutations and the non-naturally occurring C538S mutation. 6. The antibody of claim 1, wherein the RBD polypeptide is composed of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1 and includes K417T, E484K, N501Y naturally occurring mutations and C538S non-naturally occurring mutations. 7. The antibody of claim 1, wherein the RBD polypeptide is composed of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1 and includes K417N, E484Q, N501Y naturally occurring mutations and non-naturally occurring mutations C538S mutation. 8. The antibody of claim 1, wherein the RBD polypeptide is composed of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1 and includes K417T, E484Q, N501Y naturally occurring mutations and non-naturally occurring mutations C538S mutation. 9. The antibody of claim 1, wherein the RBD polypeptide is composed of amino acids ranging from the amino acid residue at position 319 to the amino acid residue at position 541 in SEQ ID NO: 1, and includes K417N, L452R, T478K, E484K, N501Y naturally occurring mutations and non-naturally occurring mutations C538S mutation. 10. The antibody of claim 1, wherein the RBD polypeptide consists of amino acids ranging from amino acid residue 319 to amino acid residue 541 in SEQ ID NO: 1 and comprising K417N , L452R, T478K, E484Q, N501Y naturally occurring mutations and C538S non-naturally occurring mutations. 11. The antibody of claim 1, wherein the heavy chain of the antibody is coupled or fused to the RBD polypeptide. 12. The antibody of claim 1, wherein the light chain of the antibody is coupled or fused to the RBD polypeptide. 13. The antibody of claim 1, wherein both the heavy and light chains of the antibody are coupled or fused to the RBD polypeptide. 14. The antibody of claim 1, wherein the heavy chain is fused or coupled to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 in SEQ ID NO: 1 to the amino acid residue at position 541 and comprising the C538S mutation, and the light chain is coupled or fused to the RBD polypeptide consisting of amino acids ranging from position 319 in SEQ ID NO: 1 to the amino acid residue at position 541, and includes the K417N, E484K, N501Y, and C538S mutations. 15. The antibody of claim 1, wherein the light chain is fused or coupled to an RBD polypeptide consisting of amino acids ranging from amino acid residue 319 in SEQ ID NO: 1 to the amino acid residue at position 541 and comprising the C538S mutation, and the heavy chain is coupled or fused to an RBD polypeptide consisting of amino acids ranging from position 319 in SEQ ID NO: 1 amino acid residue to amino acid residue 541, and includes the K417N, E484K, N501Y, and C538S mutations. 16. The antibody according to claim 1, which is an IgG antibody, preferably an IgG1 or IgG4 antibody, or even more preferably an IgG4 antibody. 17. The antibody according to claim 1, which is a chimeric antibody, in particular a chimeric mouse/human antibody or a humanized antibody. 18. The antibody of claim 1, selected from the group consisting of antibodies that specifically bind to: DC immune receptor (DCIR), MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14 , CD15, CD16, CD19, CD20, CD29, CD31, CD40, CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6 , CD40, BDCA-2, MARCO, DEC-205, mannose receptor, langerhansin, DECTIN-1, B7-1, B7-2, IFN-? receptors and IL-2 receptors, ICAM-1, Fey receptors, LOX-1 and ASPGR. 19. The antibody of claim 1, which is specific for CD40. 20. The antibody of claim 19, derived from -12E12 antibody and contains: o A heavy chain comprising the complementarity determining regions CDR1H, CDR2H and CDR3H, the CDR1H having the amino acid sequence GTFFSDYYMY (SEQ ID NO: 2), the CDR2H having the amino acid sequence YINSGGGSTYYPDTVKG (SEQ ID NO: 3) and the CDR3H having the amino acid sequence RGLPFHAMDY(SEQ ID NO:4), o and a light chain comprising the complementarity determining regions CDR1L, CDR2L and CDR3L, the CDR1L having the amino acid sequence SASQGISNYLN (SEQ ID NO:5), the CDR2L having the amino acid sequence YTSILHS (SEQ ID NO:6) and the CDR3L having the amino acid sequence Sequence QQFNKLPPT (SEQ ID NO:7), -or derived from 11B6 antibody and containing: o A heavy chain comprising the complementarity determining regions CDR1H, CDR2H and CDR3H having the amino acid sequence GYSFTGYYMH (SEQ ID NO:8), CDR2H having the amino acid sequence RINPYNGATSYNQNFKD (SEQ ID NO:9) and CDR3H having the amino acid sequence EDYVY (SEQ ID NO:10), and o A light chain comprising the complementarity determining regions CDR1L, CDR2L and CDR3L, the CDR1L having the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO: 11), the CDR2L having the amino acid sequence KVSNRFS (SEQ ID NO: 12) and the CDR3L having the amino acid sequence SQSTHVPWT(SEQ ID NO:13); -or derived from 12B4 antibody and containing: o A heavy chain comprising complementarity determining regions CDR1H, CDR2H and CDR3H, the CDR1H having the amino acid sequence GYTFTDYVLH (SEQ ID NO: 14), the CDR2H having the amino acid sequence YINPYNDGTKYNEKFKG (SEQ ID NO: 15) and the CDR3H having the amino acid sequence GYPAYSGYAMDY (SEQ ID NO:16), and o A light chain comprising the complementarity determining regions CDR1L, CDR2L and CDR3L having the amino acid sequence RASQDISNYLN (SEQ ID NO: 17), CDR2L having the amino acid sequence YTSRLHS (SEQ ID NO: 18) and CDR3L having the amino acid sequence HHGNTLPWT (SEQ ID NO :19). 21. The antibody of claim 19, wherein the anti-CD40 antibody is selected from the group consisting of mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6 selected as described in Table A. 22. The antibody of claim 19, which is a CD40 agonist antibody. 23. The antibody of claim 22, wherein the heavy or light chain of the CD40 agonist antibody (i.e., the chain that is not coupled to or fused to the RBD polypeptide) is coupled or fused to the CD40 binding of CD40L domain (SEQ ID NO:47). 24. The antibody of claim 23, wherein the CD40 binding domain of CD40L is fused to the C-terminus of the light or heavy chain of the CD40 agonist antibody, optionally via a linker, preferably a FlexV1 linker. 25. The antibody of claim 23, wherein the heavy chain of the antibody is fused or coupled to the RBD polypeptide and the light chain is coupled or fused to the CD40 binding domain of CD40L (SEQ ID NO: 44) . 26. The antibody of claim 1, which is specific for langerhansin. 27. The antibody of claim 26, comprising – a heavy chain containing the complementarity determining regions CDR1H, CDR2H and CDR3H of the 15B10 antibody and a light chain containing the complementarity determining regions CDR1L, CDR2L and CDR3L of the 15B10 antibody, or – a heavy chain containing the complementarity determining regions CDR1H, CDR2H and CDR3H of the 2G3 antibody and a light chain containing the complementarity determining regions CDR1L, CDR2L and CDR3L of the 2G3 antibody, or - A heavy chain containing the complementarity determining regions CDR1H, CDR2H and CDR3H of the 4C7 antibody, and a light chain containing the complementarity determining regions CDR1L, CDR2L and CDR3L of the 4C7 antibody. 28. The antibody of claim 26, selected from the group consisting of selected mAb7, mAb8, mAb9, mAb10, mAb11 and mAb12 as described in Table B. 29. The antibody of claim 1, wherein the heavy chain and/or the light chain is fused to the RBD polypeptide through a linker selected from FlexV1, fl, f2, f3 and f4. 30. The antibody of claim 1, comprising i) a heavy chain fused to the RBD polypeptide to form a fusion protein as shown in SEQ ID NO: 42, and ii) a light chain as shown in SEQ ID NO: 43. chain. 31. The antibody of claim 1, comprising i) a heavy chain fused to the RBD polypeptide to form a fusion protein as set forth in SEQ ID NO: 42, and ii) fused to the RBD polypeptide to form a fusion protein as SEQ ID NO: 42. The light chain of the fusion protein shown in ID NO:44. 32. The antibody of claim 1, comprising i) a heavy chain fused to the RBD polypeptide to form a fusion protein as set forth in SEQ ID NO: 45, and ii) fused to the RBD polypeptide to form a fusion protein as SEQ ID NO: 45. The light chain of the fusion protein shown in ID NO: 46. 33. The antibody of claim 1, comprising i) a heavy chain fused to the RBD polypeptide to form a fusion protein as set forth in SEQ ID NO: 42, and ii) fused to the RBD polypeptide to form a fusion protein as SEQ ID NO: 42. The light chain of the fusion protein shown in ID NO: 48. 34. The antibody of claim 1, comprising i) a heavy chain fused to the RBD polypeptide to form a fusion protein as set forth in SEQ ID NO: 49, and ii) fused to the RBD polypeptide to form a fusion protein as SEQ ID NO: 49. The light chain of the fusion protein shown in ID NO: 46. 35. The antibody of claim 1, comprising i) a heavy chain fused to the RBD polypeptide to form a fusion protein as set forth in SEQ ID NO: 42, and ii) fused to the RBD polypeptide to form a fusion protein as SEQ ID NO: 42. The light chain of the fusion protein shown in ID NO:50. 36. The antibody of claim 1, comprising i) a heavy chain fused to the RBD polypeptide to form a fusion protein as set forth in SEQ ID NO: 51, and ii) fused to the RBD polypeptide to form a fusion protein as SEQ ID NO: 51. The light chain of the fusion protein shown in ID NO: 46. 37. The antibody of claim 1, comprising i) a heavy chain fused to the RBD polypeptide to form a fusion protein as set forth in SEQ ID NO: 42, and ii) fused to the RBD polypeptide to form a fusion protein as SEQ ID NO: 42. The light chain of the fusion protein shown in ID NO:52. 38. The antibody of claim 1, comprising i) a heavy chain fused to the RBD polypeptide to form a fusion protein as set forth in SEQ ID NO: 53, and ii) fused to the RBD polypeptide to form a fusion protein as SEQ ID NO: 53. The light chain of the fusion protein shown in ID NO: 46. 39. A nucleic acid encoding the heavy chain and/or the light chain of the antibody of claim 1. 40. A vector comprising the nucleic acid of claim 37. 41. A host cell that has been transfected, infected or transformed with the nucleic acid of claim 39 and/or the vector of claim 40. 42. A vaccine composition comprising the antibody of claim 1. 43. A method of vaccinating an anti-SARS-Cov 2 vaccine to a subject in need thereof, comprising administering a therapeutically effective amount of the antibody of claim 1.