CN110903394B - Polypeptide capable of binding CD4 and application thereof - Google Patents

Polypeptide capable of binding CD4 and application thereof Download PDF

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CN110903394B
CN110903394B CN201911373825.7A CN201911373825A CN110903394B CN 110903394 B CN110903394 B CN 110903394B CN 201911373825 A CN201911373825 A CN 201911373825A CN 110903394 B CN110903394 B CN 110903394B
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CN110903394A (en
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吴喜林
吴稚伟
朱林静
袁换云
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Yuandaolong Suzhou Medical Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2812—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The invention relates to a polypeptide capable of binding CD4, which comprises 3 complementarity determining regions CDR1-3, wherein the sequence of CDR1 is or comprises one of the sequences shown in SEQ ID NO. 1-63, the sequence of CDR2 is or comprises one of the sequences shown in SEQ ID NO. 64-131, and the sequence of CDR3 is or comprises one of the sequences shown in SEQ ID NO. 132-205. According to the invention, through preparing CD4 protein, utilizing a platform technology of phage library display of nano monoclonal antibody and the like, nano antibody VHH specifically combined with CD4 is screened, CDR sequence is identified, and humanized antibody is constructed; and using the evaluation antibody for the efficacy of treating HIV infection. The invention provides a potential nano-antibody new drug, a double-target antibody new drug, a gene therapy new drug, CAR cell therapy and the like for clinical treatment of HIV infection and CD4 lymphoma by utilizing the nano-antibody combined with CD 4.

Description

Polypeptide capable of binding CD4 and application thereof
Technical Field
The invention relates to the field of biomedicine. More particularly, it relates to a polypeptide capable of binding to CD4, and also relates to the application of said polypeptide in the therapeutic medicine for HIV infection or CD4 positive T cell tumor.
Background
CD4 is an important receptor molecule expressed on the surface of T cells, but is also a target for viruses such as HIV to attack T cells. HIV is reported to affect viral entry into cells primarily through contact of the envelope surface glycoprotein gp120 with specific receptors (e.g., CD4) on the surface of host cells. Inhibiting the binding of gp120 and CD4 effectively inhibits HIV-1 infection of host cells. In addition, tumors also express CD4 due to T cells. Thus, anti-CD 4 antibodies targeted against CD4 may be considered for the treatment of HIV-infected or CD 4-positive T cell tumors.
In 1993, a novel natural antibody derived from camelidae was found. The antibody naturally lacks a light chain and consists only of a heavy chain comprising two constant regions (CH2 and CH3), a hinge region and a heavy chain Variable region (VHH, i.e., antigen binding site) with a relative molecular mass of about 13KDa, which is only 1/10 of conventional antibodies, and with a molecular height and diameter at the nanometer level, is the smallest functional antibody fragment currently available, and thus is also referred to as Nanobody (Nb). Because the nano monoclonal antibody has the characteristics of high stability (not degraded at 90 ℃), high affinity, homology of more than 80 percent with a human antibody, low toxicity and immunogenicity and the like, the nano monoclonal antibody is widely applied to the research and development of immunodiagnosis kits, the research and development of imaging, and the research and development of antibody drugs aiming at the fields of tumors, inflammations, infectious diseases, nervous system diseases and the like.
The antibody capable of binding CD4 is expected to be screened out by a new technical means, and a potential new medicine is provided for preventing and treating CD4 related diseases such as HIV infection or CD4 positive T cell tumor and the like.
Disclosure of Invention
The camel source nanometer monoclonal antibody and the VHH thereof are obtained by immunizing camel with antigen and are used for treating HIV infected patients. Based on these studies, the present invention provides a polypeptide that binds to CD4, comprising 3 CDRs 1-3, wherein the CDR1 sequence is or comprises one of the sequences shown in SEQ ID NOS 1-63, the CDR2 sequence is or comprises one of the sequences shown in SEQ ID NOS 64-131, and the CDR3 sequence is or comprises one of the sequences shown in SEQ ID NO 132-205.
In a specific embodiment, the polypeptide further comprises 4 framework regions FR1-4, said FR1-4 being staggered with respect to said CDR 1-3. For example, the FR1-4 sequence may be designed as shown in SEQ ID NO:206-209 (sources of alpaca), although the scope of the invention is not limited in this respect. The specific recognition and binding ability of an antibody is mainly determined by the CDR region sequences, and the FR sequences have little influence and can be designed according to species, which is well known in the art. For example, FR region sequences of human origin (e.g., SEQ ID NO:210-213), murine origin, or camelid origin may be designed to link the above CDRs, thereby creating a polypeptide or domain that binds CD 4.
In a preferred embodiment, the polypeptide is a monoclonal antibody.
In a preferred embodiment, the polypeptide is VHH.
In a preferred embodiment, the polypeptide is a VHH of camelid origin or a humanized VHH.
In one embodiment, the CDR sequences of the polypeptides are as follows:
I) the sequence of CD2 is SEQ ID NO 67, the sequence of CD3 is SEQ ID NO 135; and is
II) the sequence of CD1 is SEQ ID NO 4 or 5.
In one embodiment, the CDR sequences of the polypeptides are as follows:
I) the sequence of CD2 is SEQ ID NO 67, the sequence of CD3 is SEQ ID NO 135; and is
II) the sequence of CD1 is SEQ ID NO 4 or 5.
In one embodiment, the CDR sequences of the polypeptides are as follows:
I) the sequence of CD1 is GFIFXNYA, wherein X represents glycine or serine; and is
II) the sequence of CD2 is selected from SEQ ID NO: 69-71; and is
III) the sequence of CD3 is selected from the group consisting of SEQ ID NO: 137-139.
In one embodiment, the CDR sequences of the polypeptides are as follows:
I) the sequence of CD1 is GGSITTNXYY, wherein X represents aspartic acid, serine or tyrosine; and is
II) the sequence of CD2 is selected from SEQ ID NO 82-87; and is
III) the sequence of CD3 is selected from the group consisting of SEQ ID NO: 150-155.
Preferably, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO 19, the sequence of CDR2 is SEQ ID NO 82, and the sequence of CDR3 is SEQ ID NO 150; or
The sequence of CDR1 is SEQ ID NO 20, the sequence of CDR2 is SEQ ID NO 83, and the sequence of CDR3 is SEQ ID NO 151; or
The sequence of CDR1 is SEQ ID NO 20, the sequence of CDR2 is SEQ ID NO 84, and the sequence of CDR3 is SEQ ID NO 152; or
The sequence of CDR1 is SEQ ID NO 21, the sequence of CDR2 is SEQ ID NO 85, and the sequence of CDR3 is SEQ ID NO 153; or
The sequence of CDR1 is SEQ ID NO 21, the sequence of CDR2 is SEQ ID NO 86, and the sequence of CDR3 is SEQ ID NO 154; or
The sequence of CDR1 is SEQ ID NO:21, the sequence of CDR2 is SEQ ID NO:87, and the sequence of CDR3 is SEQ ID NO: 155.
In one embodiment, the CDR sequences of the polypeptides are as follows:
I) the sequence of CD1 is selected from SEQ ID NO 31-32; and is
II) the sequence of CD2 is selected from the group consisting of SEQ ID NO 97-100; and is
III) the sequence of CD3 is selected from the group consisting of SEQ ID NO 139, 165-170.
Preferably, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO 31, the sequence of CDR2 is SEQ ID NO 97, and the sequence of CDR3 is SEQ ID NO 139; or
The sequence of CDR1 is SEQ ID NO 31, the sequence of CDR2 is SEQ ID NO 98 and the sequence of CDR3 is SEQ ID NO 139; or
The sequence of CDR1 is SEQ ID NO 31, the sequence of CDR2 is SEQ ID NO 97, and the sequence of CDR3 is SEQ ID NO 165; or
The sequence of CDR1 is SEQ ID NO. 32, the sequence of CDR2 is SEQ ID NO. 99, and the sequence of CDR3 is SEQ ID NO. 166; or
The sequence of CDR1 is SEQ ID NO:32, the sequence of CDR2 is SEQ ID NO:100, and the sequence of CDR3 is SEQ ID NO: 167; or
The sequence of CDR1 is SEQ ID NO 32, the sequence of CDR2 is SEQ ID NO 97, and the sequence of CDR3 is SEQ ID NO 168; or
The sequence of CDR1 is SEQ ID NO. 32, the sequence of CDR2 is SEQ ID NO. 99, and the sequence of CDR3 is SEQ ID NO. 169; or
The sequence of CDR1 is SEQ ID NO:32, the sequence of CDR2 is SEQ ID NO:97, and the sequence of CDR3 is SEQ ID NO: 170.
In one embodiment, the CDR sequences of the polypeptides are as follows:
I) the sequence of CD1 is selected from SEQ ID NO: 39-45; and is
II) the sequence of CD2 is selected from the group consisting of SEQ ID NO: 107-113; and is
III) the sequence of CD3 is selected from the group consisting of SEQ ID NO 158, 177-187.
Preferably, the CDR sequences of the polypeptides are as follows:
the sequence of CDR1 is SEQ ID NO:39, the sequence of CDR2 is SEQ ID NO:107, and the sequence of CDR3 is SEQ ID NO: 177; or
The sequence of CDR1 is SEQ ID NO 40, the sequence of CDR2 is SEQ ID NO 108, and the sequence of CDR3 is SEQ ID NO 158; or
The sequence of CDR1 is SEQ ID NO 40, the sequence of CDR2 is SEQ ID NO 109, and the sequence of CDR3 is SEQ ID NO 178; or
The sequence of CDR1 is SEQ ID NO 41, the sequence of CDR2 is SEQ ID NO 110, and the sequence of CDR3 is SEQ ID NO 179; or
The sequence of CDR1 is SEQ ID NO 42, the sequence of CDR2 is SEQ ID NO 110, and the sequence of CDR3 is SEQ ID NO 180; or
The sequence of CDR1 is SEQ ID NO 43, the sequence of CDR2 is SEQ ID NO 110, and the sequence of CDR3 is SEQ ID NO 181; or
The sequence of CDR1 is SEQ ID NO 43, the sequence of CDR2 is SEQ ID NO 110, and the sequence of CDR3 is SEQ ID NO 182; or
The sequence of CDR1 is SEQ ID NO 43, the sequence of CDR2 is SEQ ID NO 111, and the sequence of CDR3 is SEQ ID NO 183; or
The sequence of CDR1 is SEQ ID NO 43, the sequence of CDR2 is SEQ ID NO 110, and the sequence of CDR3 is SEQ ID NO 180; or
The sequence of CDR1 is SEQ ID NO 43, the sequence of CDR2 is SEQ ID NO 110, and the sequence of CDR3 is SEQ ID NO 179; or
The sequence of CDR1 is SEQ ID NO 43, the sequence of CDR2 is SEQ ID NO 112, and the sequence of CDR3 is SEQ ID NO 184; or
The sequence of CDR1 is SEQ ID NO 43, the sequence of CDR2 is SEQ ID NO 110, and the sequence of CDR3 is SEQ ID NO 158; or
The sequence of CDR1 is SEQ ID NO 43, the sequence of CDR2 is SEQ ID NO 110 and the sequence of CDR3 is SEQ ID NO 185; or
The sequence of CDR1 is SEQ ID NO 43, the sequence of CDR2 is SEQ ID NO 111, and the sequence of CDR3 is SEQ ID NO 186; or
The sequence of CDR1 is SEQ ID NO 43, the sequence of CDR2 is SEQ ID NO 111, and the sequence of CDR3 is SEQ ID NO 179; or
The sequence of CDR1 is SEQ ID NO 43, the sequence of CDR2 is SEQ ID NO 110, and the sequence of CDR3 is SEQ ID NO 187; or
The sequence of CDR1 is SEQ ID NO. 44, the sequence of CDR2 is SEQ ID NO. 113, and the sequence of CDR3 is SEQ ID NO. 183; or
The sequence of CDR1 is SEQ ID NO 44, the sequence of CDR2 is SEQ ID NO 110, and the sequence of CDR3 is SEQ ID NO 183; or
The sequence of CDR1 is SEQ ID NO 45, the sequence of CDR2 is SEQ ID NO 110, and the sequence of CDR3 is SEQ ID NO 183.
The invention also provides application of the polypeptide in preparing a CD4 detection agent.
The invention also provides the application of the polypeptide in preparing a therapeutic drug for HIV or CD4 positive T cell tumor.
The invention also provides application of the polypeptide in preparing CAR-T therapeutic agents.
The present invention also provides the nucleic acid encoding sequence of the polypeptide.
In one embodiment, the nucleic acid coding sequence is a DNA coding sequence or an RNA coding sequence.
In a specific embodiment, the nucleic acid coding sequence is present in a gene expression cassette.
The invention also provides the application of the nucleic acid coding sequence in preparing a medicine for treating HIV infection or CD4 positive T cell tumor.
The invention carries out nano antibody drug development aiming at HIV, and by preparing CD4 protein, immunizing a bimodal camel, utilizing a platform technology of phage library display nano monoclonal antibody and the like, nano antibody VHH specifically combined with CD4 is screened, CDR sequence is identified, and humanized VHH-huFc1 is constructed; meanwhile, the efficacy of 4NB in the treatment of HIV infection was evaluated by a pseudovirus neutralization experiment. The invention provides a potential nano-antibody new drug, a double-target antibody new drug, a gene therapy new drug, CAR cell therapy and the like for clinical treatment of HIV infection and CD4 lymphoma by utilizing the nano-antibody combined with CD 4.
Drawings
FIG. 1 is a graph showing the antiserum titer test curves of CD4 after one week of 3rd and 4 th immunization of alpaca;
FIG. 2 is a graph of antiserum at different dilutions one week after the 4 th immunization of a camel inhibiting in vitro infection of ghost cells by HIV pseudovirus, with preimmune serum as a control;
FIG. 3 is an electrophoretogram of PCR products amplified using a CD4-VHH phage antibody library as a template;
FIG. 4 shows the panning identification of the CD4-VHH phage antibody library, wherein A is the ELISA detection statistical chart after phage library panning against CD4 protein; b is the second wheel (2)nd) And a third wheel (3)rd) Selecting 40 and 46 clones from the panned phage antibody library respectively to carry out phage ELISA detection statistical chart;
FIG. 5 is a statistical ELISA assay for prokaryotically expressed VHH antibodies, each dot representing a clone, with OD450 for CD 4/OD 450 for the blank on the ordinate, a positive ratio greater than 5.0 being defined;
FIG. 6 is a statistical chart of experiments with neutralization of HIV pseudoviral infection by positive VHH antibodies, one dot representing one clone and the Y-axis relative inhibition for different viruses.
Detailed Description
1. Preparation of immunogens
According to the sequence and gene sequence information of the CD4 protein, the polypeptide CD4 capable of effectively inducing camel to generate specific antibodies aiming at the CD4 protein is analyzed and designed, and His-tag (CD4-His) or rabbit Fc (CD4-rFc) is connected at the C terminal for subsequent purification and detection.
2. Preparation of alpaca immune and antiserum
Priming alpacas with an emulsified mixture of 250 μ g of CD4-rFc protein and 250 μ l of Freund's complete adjuvant, boosting 3 times with CD4-rFc protein and 250 μ l of Freund's incomplete adjuvant on days 14, 28 and 42, and collecting blood to detect antiserum titer 1 week after 2nd and 3rd immunization; after 1 week of the 4 th immunization, 200ml of blood was collected for the construction of phage antibody library.
Antiserum titers were determined by ELISA, using CD4-his protein at a concentration of 0.5. mu.g/mlTest plates were added to each well with a gradient of diluted antiserum or purified antibody 100 μ l (control pre-immune camel serum), incubated for 1.5h at 37 ℃ and washed 2 times with 1: 10000 diluted second antibody of horse radish peroxidase labeled Goat anti-Llama IgG (H + L) is incubated for 1H at 37 ℃, after washing for 4-6 times, 100 mu L of TMB substrate is added, incubation is carried out for 10min at 37 ℃, and 50 mu L of 0.2M H is added2SO4The reaction was stopped and the OD450 nm was measured. ELISA assay serum titers were specified at the highest dilution of OD450 above 2.1-fold of blank and greater than 0.2.
The results are shown in FIG. 1, and the antiserum titers of 3 and 4 of the mice were 3.28X 10, respectively6And 9.84X 106. It can be seen that the antigen can induce camels to produce high titers of antiserum specific for the CD4 protein.
To further verify whether this high titer camel antiserum was effective in preventing HIV viral infection, neutralization experiments for viral infection were performed. Antiserum and preimmune serum with different dilution concentrations are respectively incubated with HIV virus for 60min, then transferred to ghost cells, and after 48h, the virus load is detected by a Glo Max chemiluminescence enzyme-linked immunosorbent assay (Promega) instrument and the neutralization effect is calculated. The results of the neutralization experiments showed that CD 4-induced antisera inhibited 90% of HIV infection by more than 540-fold dilution of ID90 (fig. 2). Taken together, CD4 induced high titers of antisera that were also capable of inhibiting HIV pseudovirus infection with high efficacy.
Construction and panning of VHH phage library
Collecting 200ml of camel peripheral blood after immunization, separating by using lymphocyte separation liquid (GE Ficoll-Paque Plus) to obtain camel PBMC, extracting RNA according to a TRIzol operation manual, inverting by using oligo (dT) into cDNA, cloning the camel VHH gene to phagemid plasmid by using techniques such as primer amplification, molecular cloning and the like, and transforming TG1 bacteria to obtain the VHH phage library. To further identify whether the CD4-VHH phage library was successfully constructed, the VHH gene of immune CD4 camelid was amplified by PCR, and it was shown that the band of interest was 500bp, with the expected size (FIG. 3), indicating that the antibody library of CD4-VHH phage contains VHH gene. 50 clones were selected for sequencing, and the sequencing results showed thatColumns do not have completely identical repeated sequences; the alignment results show that the most of the different sequences are in the CDR binding region. Through detection, the library capacity of the constructed CD4-VHH phage antibody library is 1.6 multiplied by 109The positive rate is 100%, the sequence Diversity (Diversity) is 100%, and the effective insertion rate (In frame rate) is greater than 95%.
The phage antibody library was recovered from VHH-phagemid transformed bacteria with the help of M13KO7 helper phage and precipitated with PEG/NaCl. The phage antibody library was enriched three times with CD4-His protein coated with 50. mu.g/ml. And (3) carrying out elution, transformation, plate coating and monoclonal picking on the enriched phage, carrying out binding identification on the phage and CD4 protein ELISA, sequencing the clone with the binding reading value of more than 1.0, cloning to an expression vector phv3, and transfecting 293T cells to express to produce the nano monoclonal antibody.
The panned library was tested for binding to CD4 protein. The phage ELISA results showed that the read values of the binding between the CD4-VHH phage library and the CD4 protein before enrichment were 1.71, and the read values of the phage library after one, two and three rounds of enrichment were 2.5, 3.1 and 3.1 respectively (FIG. 4A). To further verify the positive phage rate of binding to CD4-VHH protein in the enriched library, 40 and 46 clones were selected from each of the 2nd and 3rd round enriched libraries for single phage ELISA detection. The results showed that 57.5% of individual phage clones were positive in the library round 2 and 82.6% of phage clones were positive in the library round 3, and the mean reading for binding was around 3.0 (FIG. 4B), and that the high binding CD4-VHH phage library was successfully enriched by CD4 protein panning.
Construction of VHH prokaryotic expression library and VHH expression
PCR amplification of the enriched 2nd-CD4-VHH and 3rd-CD4-VHH phage antibody libraries after the two and three rounds of panning described above; obtaining and purifying all VHH gene fragments in an antibody library, cloning the VHH gene fragments to a prokaryotic expression vector, converting an SS320 strain, and constructing a prokaryotic expression antibody library of the VHH; and (3) coating a plate with the prokaryotic expression antibody library, culturing overnight, randomly selecting 1248 monoclonal colonies the next day, inducing expression of an antibody supernatant by using IPTG (isopropyl-beta-thiogalactoside), and performing ELISA binding detection on the antibody supernatant and CD4 protein.
The results show that there was bacterial supernatant that bound CD4 protein while not binding to the blank, and that CD4 bound reads/blank reads greater than 5.0 (figure 5 and table 1). Sequencing and aligning the sequences, and removing repeated sequences to finally obtain 273 VHH antibody sequences. Further experiments demonstrated that both the 273 VHH antibodies and the CDRs derived from the VHH antibodies can specifically bind to HIV pseudoviruses.
Table 1274 binding values of VHH antibodies to CD4 protein and sequences thereof
Figure GDA0003075598410000091
Figure GDA0003075598410000101
Figure GDA0003075598410000111
Figure GDA0003075598410000121
Figure GDA0003075598410000131
Figure GDA0003075598410000141
Figure GDA0003075598410000151
Eukaryotic expression of VHH-huFc (SNB)
Through molecular cloning technology, 20 nanometer monoclonal antibody VHH genes are selected to be fused with human Fc genes and inserted into a pCDNA3.4 eukaryotic expression vector to construct and form an Nb-huFc-pCDNA3.4 expression plasmid. The constructed Nb-huFc-pCDNA3.4 is transfected into 293T cells to express and produce Nb-huFc (4 NB). Cell supernatants were collected and assayed by ELISA and some of the antibodies had good binding capacity (FIG. 6). The respective sequence numbers of the constructed humanized antibodies are shown in Table 2.
TABLE 2 original antibody numbering and CDR sequences corresponding to humanized antibody 4NB
Figure GDA0003075598410000152
6.4NB blocks HIV infection of ghost cells
The supernatants from the 20 cells were selected for in vitro neutralization. Cell supernatants were removed in 20. mu.l, combined with JRFL virus in 5% CO2Incubated at 37 ℃ for 1 hour, and 1.0X10 added4Ghost cells, 5% CO2After incubation at 37 ℃ for 48 hours in an incubator, the cell supernatant was removed, 100. mu.l/well Glolysis buffer was added (previously returned to room temperature), the plate was gently shaken to lyse the cells, 50. mu.l/well of the cell lysate was transferred to a 96-well white fluorescent microplate reader (Costar), 50. mu.l/well Brite-Glo luciferase substrate was added (previously returned to room temperature), vortexed, mixed well, and immediately placed in a Glo Max chemiluminescent microplate reader (Promega) for detection. Neutralizing titer (ID)50Or ND50) Expressed as a multiple of the dilution at 50% inhibition.
The results are shown in fig. 6, and all 4NB01, 4NB06, 4NB13, 4NB15 and 4NB02 well inhibited HIV infection, and the inhibition rates were 83%, 81%, 72%, 74% and 66%, respectively.
From the above experimental results, it is clear that the VHH antibody and humanized forms thereof of the present invention can specifically recognize and bind to CD4, and block the binding of the ligand of CD4 to CD4, thereby inhibiting diseases associated with the CD4 pathway, such as HIV infection, CD 4T cell tumor, and the like. Since these VHHs are able to recognize CD4 molecules on the cell surface, these antibody sequences can also be applied to CAR (Chimeric Antigen Receptor, consisting of VHH sequence fused to third or fourth generation CD28-4-1BB-CD3zeta molecule sequence) cells for the treatment of tumors or HIV infection. In addition, since these antibodies recognize CD4 molecules on the cell surface, VHH can be used for ADC (Antibody-drug conjugate) therapy by coupling drugs or for Antibody-dependent molecular imaging diagnosis by coupling isotopes, and the like. Nucleic acids encoding the VHHs described above may also be loaded into AAV systems for therapeutic use.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Source daolong (Suzhou) medical science and technology, Inc
<120> polypeptide capable of binding to CD4 and use thereof
<160> 213
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Glu Ser Phe Ser Asp Phe Tyr Ala
1 5
<210> 2
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Glu Ser Ser Phe Ser Ile Asn Asn
1 5
<210> 3
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Gly Asp Ser Ile Ser Thr Gly Leu Tyr Tyr
1 5 10
<210> 4
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Gly Asp Ser Ile Thr Ala Gly His Tyr Tyr
1 5 10
<210> 5
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Gly Asp Ser Ile Thr Ala Gly Tyr Tyr Tyr
1 5 10
<210> 6
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Gly Phe Phe Phe Ser Leu His Ser
1 5
<210> 7
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Gly Phe Ile Phe Gly Asn Tyr Ala
1 5
<210> 8
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Gly Phe Ile Phe Ser Asn Tyr Ala
1 5
<210> 9
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Gly Phe Ile Phe Ser Ser His Ala
1 5
<210> 10
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Gly Phe Pro Leu Asp Thr Tyr Gly
1 5
<210> 11
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Gly Phe Arg Tyr Arg Asp Tyr Ala
1 5
<210> 12
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Gly Phe Ser Phe Val Asp Phe Ala
1 5
<210> 13
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Gly Phe Ser Ile Thr Asn His Tyr
1 5
<210> 14
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Gly Phe Thr Phe Asp Gly Tyr Gly
1 5
<210> 15
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Gly Gly Ser Phe Asp Asp Tyr Thr
1 5
<210> 16
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Gly Gly Ser Ile Ala Thr Asn Tyr Tyr Tyr
1 5 10
<210> 17
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Gly Gly Ser Ile Gly Asp Asn Thr Phe Tyr
1 5 10
<210> 18
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Gly Gly Ser Ile Thr Thr Lys Tyr Ser Tyr
1 5 10
<210> 19
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Gly Gly Ser Ile Thr Thr Asn Asp Tyr Tyr
1 5 10
<210> 20
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Gly Gly Ser Ile Thr Thr Asn Ser Tyr Tyr
1 5 10
<210> 21
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Gly Gly Ser Ile Thr Thr Asn Tyr Tyr Tyr
1 5 10
<210> 22
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Gly Gly Ser Ile Thr Thr Ser Ser Tyr Phe
1 5 10
<210> 23
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Gly Gly Ser Val Thr Thr Asn Asp
1 5
<210> 24
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Gly Gly Thr Phe Ser Asn Arg Pro
1 5
<210> 25
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Gly Gly Thr Phe Ser Arg Ser Thr
1 5
<210> 26
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Gly Ile Asn Val Asp Tyr Tyr Ala
1 5
<210> 27
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Gly Ile Ser Val Arg Gly Asn Ala
1 5
<210> 28
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Gly Leu Asp Phe Asp Asp Tyr Ala
1 5
<210> 29
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Gly Leu Ser Tyr Arg Ser Asp Ala
1 5
<210> 30
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Gly Leu Thr Phe Asp Arg Tyr Val
1 5
<210> 31
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Gly Leu Thr Phe Asn Ser Asn Asn
1 5
<210> 32
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
Gly Leu Thr Phe Ser Ser Asn Ala
1 5
<210> 33
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 33
Gly Leu Thr Phe Thr Gly Tyr Trp
1 5
<210> 34
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
Gly Leu Thr Ser Asp Asp Tyr Gly
1 5
<210> 35
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
Gly Asn Ile Phe Asn Ile Asn Arg
1 5
<210> 36
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 36
Gly Arg Ala Gly Ser Lys Phe Ser
1 5
<210> 37
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 37
Gly Arg Arg Phe Pro Asn Asp Pro
1 5
<210> 38
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 38
Gly Arg Thr Phe Ser Ile Tyr Ala
1 5
<210> 39
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 39
Gly Arg Thr Phe Ser Asn Tyr Ala
1 5
<210> 40
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 40
Gly Arg Thr Phe Ser Ser Tyr Ala
1 5
<210> 41
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 41
Gly Arg Thr Ile Ser Asn Val Ala
1 5
<210> 42
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 42
Gly Arg Thr Ile Ser Ser Ile Ala
1 5
<210> 43
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
Gly Arg Thr Ile Ser Ser Val Ala
1 5
<210> 44
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 44
Gly Arg Thr Ile Ser Thr Val Ala
1 5
<210> 45
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
Gly Arg Thr Val Ser Ser Val Ala
1 5
<210> 46
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
Gly Ser Ala Pro Ser Ser Thr Val
1 5
<210> 47
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 47
Gly Ser Asp Arg Asp Ile Asp Thr
1 5
<210> 48
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 48
Gly Ser Ile Asp Ser Ile Thr Ala
1 5
<210> 49
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 49
Gly Ser Ile Phe Gly Ile Tyr Phe
1 5
<210> 50
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 50
Gly Ser Ile Phe Arg Leu Asn Ala
1 5
<210> 51
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 51
Gly Ser Ile Leu Asn Tyr Gly Gln
1 5
<210> 52
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 52
Gly Ser Ile Tyr Arg Leu Asn Tyr
1 5
<210> 53
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 53
Gly Thr Phe Ile Ser Ile Asn Thr
1 5
<210> 54
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 54
Gly Val Asp Phe Thr Ser Gly Phe Phe Phe
1 5 10
<210> 55
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 55
Gly Val Thr Phe Asp Asp Tyr Gly
1 5
<210> 56
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 56
Gly Val Val Phe Ser Leu Gly Val
1 5
<210> 57
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 57
His Arg Phe Ser Leu Glu Tyr Tyr Ser
1 5
<210> 58
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 58
Ile Leu Asp Ser Tyr Ala
1 5
<210> 59
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 59
Lys Val Pro Tyr Ser Phe Asp Arg
1 5
<210> 60
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 60
Arg Asp Gly Val Ser Ile His Ala
1 5
<210> 61
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 61
Ser Gly Ser Ile Thr Ser Asp Phe Tyr Thr
1 5 10
<210> 62
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 62
Val Asn Lys Phe Lys Ile Asp Asp
1 5
<210> 63
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 63
Val Ser Ile Phe Ser Leu Asn Val
1 5
<210> 64
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 64
Ile Asn Lys Leu Pro Gly Ile Ile
1 5
<210> 65
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 65
Ile Thr Ser Ser Gly Asn Thr
1 5
<210> 66
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 66
Val Gly Ser Gln Tyr Ser Val Gly Gly Thr
1 5 10
<210> 67
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 67
Val Thr Tyr Ser Asp Ser Thr
1 5
<210> 68
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 68
Ile Ser Arg Asp Gly Gln Arg Thr
1 5
<210> 69
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 69
Ile Ser Asn Gly Gly Leu Ser Thr
1 5
<210> 70
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 70
Ile Ser Asn Gly Gly Phe Ala Thr
1 5
<210> 71
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 71
Ile Ser Asn Gly Gly Tyr Ser Arg
1 5
<210> 72
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 72
Ile Thr His Gly Gly Asp Lys Ser
1 5
<210> 73
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 73
Ile Asn Ala Arg Asp Gly Ser Leu
1 5
<210> 74
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 74
Ile Ala Arg Met Asp Asp Ser Thr
1 5
<210> 75
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 75
Met Asn Leu Arg Asp Gln Ser Arg
1 5
<210> 76
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 76
Ile Thr Thr Gly Asp Arg Thr
1 5
<210> 77
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 77
Ile Ser Thr Asn Gly Gly Ala Thr
1 5
<210> 78
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 78
Ile Ser Asn Ser Gly Val Ser Pro
1 5
<210> 79
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 79
Ile Asp Phe Ser Gly Ser Thr
1 5
<210> 80
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 80
Ile Ser Pro Thr Gly Arg Thr
1 5
<210> 81
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 81
Ile Gly Asn Ser Gly Ser Thr
1 5
<210> 82
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 82
Ile Gly Tyr Ser Gly Ser Thr
1 5
<210> 83
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 83
Ile Ala Tyr Ser Gly Ser Thr
1 5
<210> 84
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 84
Ile Ser Asn Ser Gly Arg Thr
1 5
<210> 85
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 85
Val Asn Tyr Ala Gly Ser Thr
1 5
<210> 86
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 86
Ile His Tyr Gly Gly Ser Thr
1 5
<210> 87
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 87
Ile Asp Tyr Ser Ala Asn Thr
1 5
<210> 88
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 88
Ile Gly Phe Val Arg Glu Ser Gly Ser Thr
1 5 10
<210> 89
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 89
Ile Ile Tyr Ser Gly Ser Thr
1 5
<210> 90
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 90
Val Ser Trp Arg Gly Val Ser Thr
1 5
<210> 91
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 91
Glu Ser Trp Ser Gly Arg Thr
1 5
<210> 92
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 92
Ile Ser Ser Arg Gly Asp Ser Thr
1 5
<210> 93
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 93
Leu Ser Pro Ser Thr Asn Thr
1 5
<210> 94
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 94
Ile Asn Ser Ser Asp Gly Lys Pro
1 5
<210> 95
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 95
Ile Ser Leu Gly Asp Arg Thr
1 5
<210> 96
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 96
Ile Gly Ser Asp Asn Asn Thr
1 5
<210> 97
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 97
Ile Arg Trp Asn Ala Asn Pro
1 5
<210> 98
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 98
Ile Arg Trp Asn Ala Asn Pro Tyr
1 5
<210> 99
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 99
Ile Arg Trp Ser Ala Asn Pro
1 5
<210> 100
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 100
Ile Arg Trp Asn Gly Asn Pro
1 5
<210> 101
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 101
Ile Asn Ser Gly Gly Gly Ser Thr
1 5
<210> 102
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 102
Phe Ser Thr Gly Asp Ala Ser Thr
1 5
<210> 103
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 103
Arg Thr Asn Gly Gly Ala Leu
1 5
<210> 104
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 104
Leu Pro Trp Asn Asp Arg Asn Pro
1 5
<210> 105
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 105
Ile Ser Gly Asn Gly Met Ser Thr
1 5
<210> 106
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 106
Ile Asn Ser Ala Ser Gly Ser Thr
1 5
<210> 107
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 107
Ile Ser Trp Ser Gly Asp Arg Thr
1 5
<210> 108
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 108
Ile Ser Arg Ser Gly Ser Arg Ala
1 5
<210> 109
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 109
Ile Ser Trp Arg Gly Gly Asn Thr
1 5
<210> 110
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 110
Ile Thr Trp Ser Gly Asp Tyr Thr
1 5
<210> 111
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 111
Ile Thr Trp Ser Gly Glu Tyr Thr
1 5
<210> 112
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 112
Ile Thr Trp Asn Gly Asp Tyr Thr
1 5
<210> 113
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 113
Ile Thr Trp Ser Gly Asn Tyr Thr
1 5
<210> 114
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 114
Ile Asn Trp Asn Gly Asp Gln Thr
1 5
<210> 115
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 115
Ile Thr Lys Gly Gly Ser Thr
1 5
<210> 116
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 116
Arg Pro Phe Gln Gly Ser Thr
1 5
<210> 117
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 117
Ile Thr Gly Ser Gly Ser Thr
1 5
<210> 118
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 118
Ile Ser Gly Asp Gly Val Thr
1 5
<210> 119
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 119
Ile Thr Ser Gly Asp Arg Thr
1 5
<210> 120
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 120
Ile Asn Gly Asp Ala Asp Val
1 5
<210> 121
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 121
Ile Thr Ser Asp Gly Asn Ala
1 5
<210> 122
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 122
Ile Thr Asn Thr Gly Ala Thr
1 5
<210> 123
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 123
Phe Ser Asp Val Asp Ser Lys Thr
1 5
<210> 124
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 124
Ile Thr Ser Gly Gly Ser Ala
1 5
<210> 125
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 125
Asn Gly Ile Asp Lys Ser Leu
1 5
<210> 126
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 126
Thr Asn Ala Asp Gly Ser Ala
1 5
<210> 127
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 127
Ile Ser Asn Gly Gly Ser Ile
1 5
<210> 128
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 128
Ile Thr Thr Asp Gly Arg Thr
1 5
<210> 129
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 129
Ile Gly Leu Ser Gly Ile Thr
1 5
<210> 130
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 130
Ile Thr Glu Gly Gly Thr Val
1 5
<210> 131
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 131
Val Thr Arg Ser Gly Pro Thr
1 5
<210> 132
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 132
Ala Val Arg Ser Phe Tyr Ser Gly Ala Asn Phe Pro Glu Trp Asn Ser
1 5 10 15
Asp Tyr Glu Tyr Asp Tyr
20
<210> 133
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 133
Lys Leu Glu Arg Gly Gly Ile Phe Leu Tyr Asp Tyr
1 5 10
<210> 134
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 134
Thr Arg Gly Asp Trp Leu Ile Asn Thr Ile Gly Arg Leu Asp Asp Phe
1 5 10 15
Asp Tyr
<210> 135
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 135
Thr Arg Gly Asp Trp Leu Ile Asn Thr Val Thr Arg Leu Asp Asp Phe
1 5 10 15
Asp Ser
<210> 136
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 136
Ala Lys Ser Lys Ser Asp Thr Tyr Tyr Thr Gly Thr Tyr Lys Ser Glu
1 5 10 15
Asn Tyr Glu Ala
20
<210> 137
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 137
Ala Lys Tyr Ser Ala Ala Trp Pro Pro Asn Asp Trp Arg Asn Tyr Asp
1 5 10 15
Ala
<210> 138
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 138
Val Lys Tyr Ser Gly Ala Trp Pro Pro Asn Asp Trp Arg Asn Tyr Asp
1 5 10 15
Ala
<210> 139
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 139
Ala Ala Ala Ala Asp Ala Tyr Tyr Arg Phe Gly Asp Tyr Phe Val Gln
1 5 10 15
Gln Ser Ser Tyr Asp Tyr
20
<210> 140
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 140
Ala Arg Trp Asn Gly Ala Gly Asp Phe Pro Gly Ser His Phe Asp Tyr
1 5 10 15
<210> 141
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 141
Ala Ala Ser Ile Ser Asn Tyr Cys Pro Leu Tyr Ile His Phe Asp Tyr
1 5 10 15
Thr Met Asp Tyr
20
<210> 142
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 142
Ala Ala Asp Gln Asn Ala Asp Cys Phe Ala Phe Glu Lys His Arg Phe
1 5 10 15
Gly Ser
<210> 143
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 143
Ala Val Arg Thr Arg Cys Ser Gly Tyr Asp Leu Gly Arg Leu Arg Tyr
1 5 10 15
Leu Glu Val
<210> 144
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 144
His Arg Pro Val Gly Ser Leu
1 5
<210> 145
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 145
Ala Lys Pro Lys Met Asp Thr Ala Tyr Pro Asp Ala Phe Ser Ser Asp
1 5 10 15
Asp Tyr Asp Ser
20
<210> 146
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 146
Ala Ala Asp Ser Tyr Arg Asn Cys Leu Asp Leu Arg Val Pro Gly Leu
1 5 10 15
Pro Trp Asp Tyr
20
<210> 147
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 147
Ala Gly Gln Gly Gly Gly Thr Leu Ala Thr Tyr Gly Ser
1 5 10
<210> 148
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 148
Ala Lys Thr Leu Ser Ser Arg Cys Ser Ser Val Glu Val
1 5 10
<210> 149
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 149
Ala Arg Met Ile Tyr Gly Ser Arg Trp Phe Asn Thr Gly Tyr Gly Met
1 5 10 15
Asp Tyr
<210> 150
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 150
Ala Arg Ile Arg Val Pro Tyr Tyr Tyr Cys Ser Gly Tyr Gly Cys Ser
1 5 10 15
Glu Gly Ala
<210> 151
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 151
Ala Arg Leu Gln Gly Ser Asn Trp Phe Pro Gly Lys Thr Tyr Glu Phe
1 5 10 15
Asp Tyr
<210> 152
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 152
Ala Gly Gly Trp Gly Gly Leu Ser Tyr Ser Asn Tyr
1 5 10
<210> 153
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 153
Ala Arg Gly Pro Ser Asn Val Val Pro Gly Thr Asn
1 5 10
<210> 154
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 154
Ala Arg Asp Ser Glu Tyr Leu Ser Ser Pro Gly Val Ala Val Ala Phe
1 5 10 15
Gly Ser
<210> 155
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 155
Ala Arg Val His Gly Asn Tyr Trp Ser Asp Arg Tyr Phe Glu Val
1 5 10 15
<210> 156
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 156
Ala Arg Ser Asp Tyr Ser Ala His His Ser Glu Asp Pro Ser Ser
1 5 10 15
<210> 157
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 157
Ala Arg Val Ala Pro Asp Arg Arg Gly Ser Met Trp Trp Asp Arg Met
1 5 10 15
Asp Tyr
<210> 158
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 158
Ala Ala Thr Thr Val Ala Gly Phe His Thr Leu Leu Val Val Thr Thr
1 5 10 15
Ser Tyr His Tyr
20
<210> 159
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 159
Val Ala Asp Arg Glu Ser Leu Arg Val Ala Pro Arg Pro Ala His Asn
1 5 10 15
Tyr Glu Phe
<210> 160
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 160
Ala Ala Thr Pro Gly Gly Pro Arg Thr Asp Gly Asp Cys Leu Asp His
1 5 10 15
Ala Arg Met Asp Leu
20
<210> 161
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 161
Asn Arg Tyr Pro Ser Thr Asp Tyr
1 5
<210> 162
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 162
Ala Ala Glu Arg Trp Ile Cys Gln Leu His Arg Pro Asp Ala Tyr Asp
1 5 10 15
Tyr
<210> 163
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 163
Ala Ala Gly Gly Gly Asn Trp Tyr Ala Leu Asp Met Asp Tyr
1 5 10
<210> 164
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 164
Gly Val Trp Leu His Thr Thr Ala Leu Ser Thr Glu Pro Met Phe Tyr
1 5 10 15
Asp Tyr
<210> 165
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 165
Ala Ala Ala Ala Asp Ala Tyr Tyr Arg Phe Gly Asp Tyr Leu Val Gln
1 5 10 15
Gln Ser Ser Tyr Asp Tyr
20
<210> 166
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 166
Ala Ala Ala Ala Asp Ala Tyr Tyr Arg Tyr Gly Asn Tyr Phe Val His
1 5 10 15
Glu Ser Ser Tyr Asp Tyr
20
<210> 167
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 167
Ala Ala Ala Ala Asp Ala Tyr Tyr Arg Tyr Gly Asn Tyr Phe Val Gln
1 5 10 15
Gln Ser Ser Tyr Asp Tyr
20
<210> 168
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 168
Ala Ser Ala Ala Asp Ala Tyr Tyr Arg Phe Gly Asn Tyr Phe Val Gln
1 5 10 15
Gln Ser Ser Tyr Asp Tyr
20
<210> 169
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 169
Ala Ala Ala Ala Asp Ala Tyr Tyr Leu Tyr Gly Ser Asp Phe Val Gln
1 5 10 15
Gln Thr Ser Tyr Asp Tyr
20
<210> 170
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 170
Ala Ala Ala Ala Asp Ala Tyr Tyr His Tyr Gly Asn Tyr Phe Val Gln
1 5 10 15
Gln Ser Ser Tyr Asp Tyr
20
<210> 171
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 171
Ala Ser Gly Tyr Gln Thr Ser Gly Tyr
1 5
<210> 172
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 172
Ala Leu Arg His Tyr Tyr His Cys Ser Ile Trp Gly Pro Gly Asp Met
1 5 10 15
Asp Tyr
<210> 173
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 173
Asn Ala Gly Asp Pro Lys Gly Asp Tyr
1 5
<210> 174
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 174
Ala Val Ala Arg Phe Gly Thr Thr Arg Pro Glu Tyr Ala Ala Glu Tyr
1 5 10 15
Glu Tyr
<210> 175
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 175
Ala Ala Asp Ile Asp Arg Gly Thr Met Asn Leu Arg Arg Arg Val Glu
1 5 10 15
Tyr Asp Tyr
<210> 176
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 176
Ala Ala Lys Pro Val Gly Arg Ala Ile Ser Gly Thr Tyr Arg Asp Pro
1 5 10 15
Asn Ala Tyr Thr Tyr
20
<210> 177
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 177
Ala Ala Asp Gly Ala Arg Gly Pro Phe Val Ile Leu Thr Val Lys Ser
1 5 10 15
Ser Tyr Asp His
20
<210> 178
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 178
Ala Ala Asp Gln Tyr Gly Arg Gly His Ser Gly Gly Tyr Glu Tyr Glu
1 5 10 15
Tyr
<210> 179
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 179
Ala Ala Asp Leu Arg Gly Gly Ser Ile Tyr Gly Thr Glu Asp Tyr Val
1 5 10 15
Tyr
<210> 180
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 180
Ala Ala Asp Leu Arg Gly Gly Ser Ile Tyr Gly Ser Gln Asp Tyr Val
1 5 10 15
Tyr
<210> 181
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 181
Ala Ala Asp Leu Arg Gly Gly Ser Ile Tyr Gly Ser Glu Asp Tyr Val
1 5 10 15
Tyr
<210> 182
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 182
Ala Ala Asp Leu Arg Gly Ser Ser Ile Tyr Gly Ser Glu Asp Tyr Val
1 5 10 15
Tyr
<210> 183
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 183
Ala Ala Asp Leu Arg Gly Gly Ser Ile Tyr Gly Thr Gln Asp Tyr Val
1 5 10 15
Tyr
<210> 184
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 184
Ala Ala Asp Leu Arg Gly Ser Ser Ile Tyr Gly Ser Gln Asp Tyr Val
1 5 10 15
Tyr
<210> 185
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 185
Ala Ala Asp Leu Arg Gly Gly Ser Ile Tyr Gly Arg Gln Asp Tyr Val
1 5 10 15
Tyr
<210> 186
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 186
Ala Ala Asp Leu Cys Gly Gly Ser Ile Tyr Gly Thr Glu Asp Tyr Val
1 5 10 15
Tyr
<210> 187
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 187
Ala Ala Asp Leu Arg Gly Gly Ser Ile Tyr Gly Arg Gln Asn Tyr Val
1 5 10 15
Tyr
<210> 188
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 188
Ala Ala Arg Ala Pro Gly Tyr Val Ser Leu Glu Arg Ser Ser Ser Thr
1 5 10 15
Tyr Asn Ser
<210> 189
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 189
Asn Ala Phe Val Ile Val Phe Gly Arg Pro Pro Tyr Asp Ser Gly Asp
1 5 10 15
Tyr
<210> 190
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 190
Glu Ala Glu Ala Tyr Gly Arg Leu Gln Phe
1 5 10
<210> 191
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 191
Asn Thr Glu Val Trp Ile Ser Asp Asp Asp Glu Val Arg Tyr Asp Tyr
1 5 10 15
<210> 192
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 192
Asn Val Lys Tyr Met Glu Arg Thr Ala Phe Thr Thr Ala Tyr Asp Tyr
1 5 10 15
<210> 193
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 193
Tyr Ala Arg Arg Trp Tyr Thr Asp Tyr
1 5
<210> 194
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 194
Asn Ile Val Ala Glu His Ile Arg Gly Ser Ala Arg Asn Tyr
1 5 10
<210> 195
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 195
Asn Ala Asn Val Arg Ile Gly Val Leu Asn Arg Arg Asp Tyr
1 5 10
<210> 196
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 196
Ala Arg Gly Asn Ser Gly Thr Gln Tyr Leu Gly Ser Asn Ala Met Asp
1 5 10 15
Ala
<210> 197
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 197
Ala Arg Ser His Thr Gln Gly Ala Thr Trp Cys Phe Ala Glu Asp Tyr
1 5 10 15
Gly Ser
<210> 198
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 198
Asn Ala His Leu Gly Tyr Ser Gly Asp Glu Tyr
1 5 10
<210> 199
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 199
Ala Ala Arg Val Pro Ser Phe Gly Ser Thr Trp Asp Pro Ala Ser Ala
1 5 10 15
His Ala Tyr Thr Tyr
20
<210> 200
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 200
Ala Thr Asp Arg Gln Phe Ser Cys Gly Ser Asp Trp His Phe Gly Met
1 5 10 15
Asn Tyr
<210> 201
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 201
His Leu Tyr Gly Ala Gly Gly Ile Leu Asp Ala
1 5 10
<210> 202
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 202
Ala Ala Gly Asn Arg Tyr Phe Asn Pro Ser Lys Ser Ser Tyr Tyr Tyr
1 5 10 15
Gly Met Asp Tyr
20
<210> 203
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 203
Ala Arg Gly Gly Glu Arg Glu Ser Pro Asn Tyr Arg Tyr Thr Arg Leu
1 5 10 15
Glu Ile
<210> 204
<211> 18
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 204
His Val Gly Gly Leu Asp Gly Asn Gly Trp Tyr Gly Asn Arg Tyr Arg
1 5 10 15
Glu His
<210> 205
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 205
Ile Ala Val Pro Ser Ser Thr Pro Tyr
1 5
<210> 206
<211> 27
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 206
Met Ala Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
20 25
<210> 207
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 207
Met Gly Trp Phe Arg Gln Ala Pro Glu Lys Glu Arg Glu Phe Val Ala
1 5 10 15
Ala
<210> 208
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 208
Asn Val Ala Asp Ser Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Arg Gln Thr Val Ser Leu Gln Leu Thr Asn Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 209
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 209
Trp Gly Pro Gly Thr Gln Val Thr Val Ser Ala Glu Pro Lys Thr Pro
1 5 10 15
Lys Pro Gln Pro
20
<210> 210
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 210
Gln Val Arg Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu
1 5 10 15
Thr Leu Arg Leu Ser Cys Thr Ala Ser
20 25
<210> 211
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 211
Met Gly Trp Tyr Arg Gln Gly Pro Gly Asn Glu Cys Glu Met Val Ala
1 5 10 15
Tyr
<210> 212
<211> 36
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 212
Ala Asp Ser Thr Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys
1 5 10 15
His Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly
20 25 30
Val Tyr Tyr Cys
35
<210> 213
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 213
Gly Gln Gly Thr Arg Val Thr Val Ser Ser
1 5 10

Claims (8)

1. A nanobody capable of binding to CD4, comprising 3 CDRs 1-3, wherein the sequences of the CDRs of the nanobody are as follows:
the sequence of CDR1 is shown in SEQ ID NO. 43, the sequence of CDR2 is shown in SEQ ID NO. 110, and the sequence of CDR3 is selected from one of SEQ ID NO. 179, 180 and 183; or
The sequence of CDR1 is shown in SEQ ID NO:39, the sequence of CDR2 is shown in SEQ ID NO:107, and the sequence of CDR3 is shown in SEQ ID NO: 177; or
The sequence of CDR1 is shown in SEQ ID NO. 31, the sequence of CDR2 is shown in SEQ ID NO. 97, and the sequence of CDR3 is shown in SEQ ID NO. 139.
2. The nanobody of claim 1, further comprising 4 framework regions FR1-4, wherein the FR1-4 is sequentially staggered from the CDR 1-3.
3. Nanobody according to claim 1, characterized in that it is of camelid or humanized VHH.
4. Use of the nanobody of any one of claims 1 to 3 for the preparation of a CD4 detection agent.
5. Use of the nanobody of any one of claims 1 to 3 for the preparation of a medicament for the treatment of HIV.
6. Use of a nanobody according to any of claims 1 to 3 for the preparation of a CAR-T therapeutic.
7. A nucleic acid encoding the nanobody of any one of claims 1 to 3.
8. Use of the nucleic acid of claim 7 for the preparation of a medicament for the treatment of HIV infection.
CN201911373825.7A 2019-12-27 2019-12-27 Polypeptide capable of binding CD4 and application thereof Active CN110903394B (en)

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CN110903394B (en) * 2019-12-27 2021-09-07 源道隆（苏州）医学科技有限公司 Polypeptide capable of binding CD4 and application thereof
CN111808193B (en) * 2020-06-30 2022-04-05 源道隆（苏州）医学科技有限公司 Nanobody capable of binding human CD38 and application thereof
EP4112640A1 (en) * 2021-06-29 2023-01-04 NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen Novel single domain antigen binding molecules and their uses
CN114213539B (en) * 2021-12-29 2023-05-16 源道隆（苏州）医学科技有限公司 Nanobody 4NB357 capable of binding CD4 and application thereof
CN114276453A (en) * 2021-12-29 2022-04-05 源道隆（苏州）医学科技有限公司 Nanobody 4NB334 capable of binding CD4 and application thereof

Citations (2)

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CN1121725A (en) * 1993-04-22 1996-05-01 Upsa实验室公司 Immunoparticles bearing monoclonal anti-CD4 antibodies and utilisation thereof
CN102649818A (en) * 2011-02-25 2012-08-29 厦门大学 CD4 protein-resistant monoclonal antibody and active fragment and application thereof

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EP2352764B1 (en) * 2008-10-14 2018-03-28 Ablynx N.V. AMINO ACID SEQUENCES TARGETING HUMAN CD4 and CXCR4, CCR5, TLR4, ALPHAV INTEGRIN, BETA3-INTEGRIN,BETA1-INTEGRIN, HUMAN ALPHA2-INTEGRIN, CD81, SR-BI, CLAUDIN-1, CLAUDIN-6 AND/OR CLAUDIN-9, RESPECTIVELY, AND NEUTRALIZING VIRAL ENTRY
CN106188292B (en) * 2015-05-05 2019-09-24 北京傲锐东源生物科技有限公司 Anti- CD4 protein monoclonal antibody and application thereof
CN110452881A (en) * 2019-07-25 2019-11-15 福建谷科生物科技有限公司 Using CD4 as the preparation and application of the antibody of target spot and CAR-T cell
CN110903394B (en) * 2019-12-27 2021-09-07 源道隆（苏州）医学科技有限公司 Polypeptide capable of binding CD4 and application thereof

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CN1121725A (en) * 1993-04-22 1996-05-01 Upsa实验室公司 Immunoparticles bearing monoclonal anti-CD4 antibodies and utilisation thereof
CN102649818A (en) * 2011-02-25 2012-08-29 厦门大学 CD4 protein-resistant monoclonal antibody and active fragment and application thereof

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