# US 10,479,990 B2 · Semi-synthetic nurse shark VNAR libraries for making and using selective binding compounds

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- Number: US 10,479,990 B2
- Document ID: US10479990B2
- Status: Granted
- Kind: B2
- Application number: 15/321,314
- Filed: 2015-06-26
- Published: 2017-07-13
- Granted: 2019-11-19
- Earliest claimed: 2014-06-26
- Assignees: Ossianix, Inc.
- Applicants: Ossianix, Inc.
- Inventors: Julien Häsler; Julia Lynn Rutkowski
- Web page: https://patentgrep.com/patent/US10479990B2

## CPC

- C12N 15/1055: {Protein x Protein interaction, e.g. two hybrid selection}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/005: {constructed by phage libraries}
- C07K 16/22: against growth factors {; against growth regulators}
- C07K 16/2875: {against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154 (against NGF C07K16/22, against TNF C07K16/241)}
- C07K 16/2881: {against CD71}
- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- C07K 2317/20: characterized by taxonomic origin
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/567: Framework region [FR]
- C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
- C07K 2317/72: Increased effector function due to an Fc-modification
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C40B 40/08: Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
- C40B 40/10: Libraries containing peptides or polypeptides, or derivatives thereof
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

The present invention relates to VNAR single chain antibodies and more particularly, to semi-synthetic VNAR libraries derived from nurse shark which may be used to identify individual clones, nucleic acid molecules and polypeptides which encode binding moieties that specifically bind to a cellular target of interest, thereby altering (e.g., antagonizing) target activity in a cell or mimicking the activity of a native molecule. The present invention thus also relates to compounds and compositions comprising a target specific VNAR binding moiety, methods for preparing them, and diagnostic and therapeutic methods of use relating to regulation, e.g., agonism or antagonism of the selected cellular target or target pathway e.g., to treat and/or prevent a pathological condition, disorder or disease in which it is beneficial to alter, e.g., agonize or augment, antagonize, reduce or eliminate the specific cellular target activity.

## Claims

### Claim 1

A nucleic acid-containing library whose members each comprise a nucleic acid encoding a polypeptide comprising a VNAR Type 2 backbone amino acid sequence comprising a semi-synthetic Type 2 VNAR framework (FW), hypervariable (HV) and complementary determining region (CDR) regions having a domain structure, from N- to C-terminal:
FW1-CDR1-FW2-HV2-FW2′-HV4-FW3-CDR3-FW4
wherein said polypeptide comprises VNAR Type 2 backbone amino acid sequence:
ARVDQTPX1X2X3(10)TKETGESLTI(20)NCVLRDX4X5CX6(30)LX7X8X9X10WYR X11K(40)SX12SX13NEX14X15IS(50)X16GGRYVETVN(60)SGSKSFSLX17I(70)NDLX18VX19DSGT(80) YRCX20V(85)-[ . . . CDR3 . . . ]-YGX21GTX22VTVNA (SEQ ID NO. 9),
wherein amino acid compositions at each position X are:
X1=Q and R, with R being present in more than 4.4% of the polypeptides;
X2=T and S, with S being present in more than 5.4% of the polypeptides;
X3=I and V, with V being present in more than 4.4% of the polypeptides;
X4=S and N
X5=N and I, with I being present in more than 3.2% of the polypeptides;
X6=A, D and E, with D being present in more than 3.2% of the polypeptides and E being present in more than 2.2% of the polypeptides;
X7=S and P, with P being present in more than 2.1% of the polypeptides;
X8=S, R, T and N, with T being present in more than 1% of the polypeptides, and N being present in more than 5.3% of the polypeptides;
X9=T and L, with L being present in more than 1.1% of the polypeptides;
X10=Y, H and L, with H being present in more than 2.1% of the polypeptides and L being present in more than 2.1% of the polypeptides;
X11=K and T, with T being present in more than 4.4% of the polypeptides;
X12=G and D, with D being present in more than 4.4% of the polypeptides;
X13=T and R, with R being present in more than 5.3% of the polypeptides;
X14=E and A, with A being present in more than 1.1% of the polypeptides;
X15=S, N and R, with R being present in more than 3.2% of the polypeptides;
X16=K and L, with L being present in more than 3.2% of the polypeptides;
X17=R and K, with K being present in more than 3.2% of the polypeptides;
X18=T and V, with V being present in more than 3.2% of the polypeptides;
X19=E and K, with K being present in more than 1.1% of the polypeptides;
X20=N and K;
X21=G and D; and
X22=V and A; and
wherein CDR3 comprises an amino acid sequence of from 7 to 25 amino acid residues,
wherein CDR3 comprises at least one cysteine residue,
wherein the last two amino acid residues of CDR3 are DV, and
wherein CDR3 is otherwise randomized compared to germline Type 2 VNAR sequences.

### Claim 2 (depends on 1)

The nucleic acid library of claim 1, wherein the CDR3 of each polypeptide is from 7 to 25, from 9 to 20 or from 11 to 18 amino acid residues in length.

### Claim 3 (depends on 2)

The nucleic acid library of claim 2, wherein together the last two amino acids of the FW3 region, the CDR3 region and the first six amino acids of the FW4 region, collectively CDR3*, are selected from any one of the sequences
(i)
NV[X]4C[X]4DVYGGGTV, NV[X]4C[X]4DVYGGGTA,
(11-mer)
NV[X]4C[X]4DVYGDGTA, NV[X]3[Z]3[X]3DVYGGGTV,
NV[X]3[Z]3[X]3DVYGGGTA, NV[X]3[Z]3[X]3DVYGDGTA,
(SEQ ID NOS. 10-15, respectively),
(ii)
NV[X]5C[X]4DVYGGGTV, NV[X]5C[X]4DVYGGGTA,
(12-mer)
NV[X]5C[X]4DVYGDGTA, NV[X]3[Z]3[X]4DVYGGGTV,
NV[X]3[Z]3[X]4DVYGGGTA, NV[X]3[Z]3[X]4DVYGDGTA,
(SEQ ID NOS. 16-21, respectively),
(iii)
NV[X]7C[X]3DVYGGGTV, NV[X]7C[X]3DVYGGGTA,
(13-mer)
NV[X]7C[X]3DVYGDGTA, NV[X]5[Z]3[X]3DVYGGGTV,
NV[X]5[Z]3[X]3DVYGGGTA, NV[X]5[Z]3[X]3DVYGDGTA,
(SEQ ID NOS. 22-27, respectively),
(iv)
NV[X]7C[X]4DVYGGGTV, NV[X]7C[X]4DVYGGGTA,
(14-mer)
NV[X]7C[X]4DVYGDGTA, NV[X]6[Z]3[X]3DVYGGGTV,
NV[X]6[Z]3[X]3DVYGGGTA, NV[X]6[Z]3[X]3DVYGDGTA,
(SEQ ID NOS. 28-33, respectively),
(v)
NV[X]7C[X]5DVYGGGTV, NV[X]7C[X]5DVYGGGTA,
(15-mer)
NV[X]7C[X]5DVYGDGTA, NV[X]6[Z]3[X]4DVYGGGTV,
NV[X]6[Z]3[X]4DVYGGGTA, NV[X]6[Z]3[X]4DVYGDGTA,
(SEQ ID NOS. 34-39, respectively),
(vi)
KV[X]7C[X]6DVYGGGTV, KV[X]7C[X]6DVYGGGTA,
(16-mer)
KV[X]7C[X]6DVYGDGTA, KV[X]3ZXZXZ[X]6DVYGGGTV,
KV[X]3ZXZXZ[X]6DVYGGGTA,
KV[X]3ZXZXZ[X]6DVYGDGTA,
(SEQ ID NOS. 40-45, respectively),
(vii)
KV[X]6C[X]8DVYGGGTV, KV[X]6C[X]8DVYGGGTA,
(17-mer)
KV[X]6C[X]8DVYGDGTA, KV[X]6[Z]3[X]6DVYGGGTV,
KV[X]6[Z]3[X]6DVYGGGTA, KV[X]6[Z]3[X]6DVYGDGTA,
(SEQ ID NOS. 46-51, respectively),
(viii)
KV[X]10C[X]5DVYGGGTV, KV[X]10C[X]5DVYGGGTA,
(18-mer)
KV[X]10C[X]5DVYGDGTA, KV[X]8[Z]3[X]5DVYGGGTV,
KV[X]8[Z]3[X]5DVYGGGTA or KV[X]8[Z]3[X]5DVYGDGTA
(SEQ ID NOS. 52-57, respectively),
and wherein each X is independently any amino acid and each Z is N, S, D, G, Y or C, provided that at least one Z is C.

### Claim 4 (depends on 3)

The nucleic acid-containing library of claim 3 wherein
FW1-CDR1-FW2-HV2-FW2′-HV4-FW3, less the last two amino acids of FW3, has an amino acid sequence of any one of molecules VNAR_1 to VNAR_10 (SEQ ID NOS. 320, 321, 319 and 322-328, respectively); and
the remaining residues of FW4 have an amino acid sequence of VTVNA (SEQ ID NO. 58).

### Claim 5 (depends on 1)

The nucleic acid library of claim 1, wherein said library is a phage display library.

### Claim 6 (depends on 1)

A method of identifying a polypeptide that binds selectively to a target molecule of interest which comprises:
a) exposing a target molecule of interest to polypeptides produced by expression of a library of claim 1; and
b) separating polypeptides that selectively bind from those that do not selectively bind the target molecule.

### Claim 7 (depends on 6)

The method of claim 6, wherein the target molecule of interest is expressed on the surface of a phage, bacterium or cell, or is attached to, tethered to or otherwise associated with a solid support.

### Claim 8 (depends on 6)

The method of claim 6 which further comprises:
c) identifying high affinity binders from the polypeptide binders, wherein high affinity binders have an affinity of from about 0.1 nM to 250 nM, 1 nM to 250 nM, 10 nM to 250 nM, 50 nM to 250 nM, 100 nM to 250 nM, 0.1 nM to 100 nM, 1 nM to 100 nM, 10 nM to 100 nM, 50 nM to 100 nM, 0.1 nM to 50 nM, 1 nM to 50 nM, 5 nM to 50 nM, 10 nM to 50 nM, 0.1 nM to 25 nM, 1 nM to 25 nM, 5 nM to 25 nM, or 10 nM to 25 nM.

### Claim 9 (depends on 6)

The method of claim 6 for identifying one or more polypeptides that selectively bind to a target molecule of interest, wherein exposing a target molecule of interest to a composition comprises:
a) contacting said target with a nucleic acid-containing library whose members each comprise a nucleic acid encoding a polypeptide comprising a VNAR Type 2 backbone amino acid sequence comprising a semi-synthetic Type 2 VNAR framework (FW), hypervariable (HV) and complementary determining region (CDR) regions having a domain structure, from N- to C-terminal:
FW1-CDR1-FW2-HV2-FW2′-HV4-FW3-CDR3-FW4
wherein said polypeptide comprises VNAR Type 2 backbone amino acid sequence:
ARVDQTPX1X2X3(10)TKETGESLTI(20)NCVLRDX4X5CX6(30)LX7X8X9X10WYR X11K(40)SX12SX13NEX14X15IS(50)X16GGRYVETVN(60)SGSKSFSLX17I(70)NDLX18VX19DSGT(80) YRCX20V(85)-[ . . . CDR3 . . . ]-YGX21GTX22VTVNA (SEQ ID NO. 9),
wherein amino acid compositions at each position X are:
X1=Q and R, with R being present in more than 4.4% of the polypeptides;
X2=T and S, with S being present in more than 5.4% of the polypeptides;
X3=I and V, with V being present in more than 4.4% of the polypeptides;
X4=S and N
X5=N and I, with I being present in more than 3.2% of the polypeptides;
X6=A, D and E, with D being present in more than 3.2% of the polypeptides and E being present in more than 2.2% of the polypeptides;
X7=S and P, with P being present in more than 2.1% of the polypeptides;
X8=S, R, T and N, with T being present in more than 1% of the polypeptides, and N being present in more than 5.3% of the polypeptides;
X9=T and L, with L being present in more than 1.1% of the polypeptides;
X10=Y, H and L, with H being present in more than 2.1% of the polypeptides and L being present in more than 2.1% of the polypeptides;
X11=K and T, with T being present in more than 4.4% of the polypeptides;
X12=G and D, with D being present in more than 4.4% of the polypeptides;
X13=T and R, with R being present in more than 5.3% of the polypeptides;
X14=E and A, with A being present in more than 1.1% of the polypeptides;
X15=S, N and R, with R being present in more than 3.2% of the polypeptides;
X16=K and L, with L being present in more than 3.2% of the polypeptides;
X17=R and K, with K being present in more than 3.2% of the polypeptides;
X18=T and V, with V being present in more than 3.2% of the polypeptides;
X19=E and K, with K being present in more than 1.1% of the polypeptides;
X20=N and K;
X21=G and D; and
X22=V and A; and
wherein CDR3 comprises an amino acid sequence of from 7 to 25 amino acid residues,
wherein CDR3 comprises at least one cysteine residue,
wherein the last two amino acid residues of CDR3 are DV,
wherein CDR3 is otherwise randomized compared to germline Type 2 VNAR sequences,
wherein said library is a phage display library; and
b) separating phage that selectively bind said target molecule from those that do not selectively bind said target molecule to produce an enriched phage library;
c) repeating steps a) and b) with said enriched phage library to produce a further enriched phage library; and
d) repeating step c) until said further enriched phage library is enriched from at least about 10- to about 106-fold or more relative to the original phage library; and
wherein separating polypeptides that selectively bind from those that do not selectively bind the target molecule is achieved by
e) plating said further enriched phage library and isolating and characterizing individual clones therefrom and thereby identifying one or more polypeptides that selectively bind to a target molecule of interest.

### Claim 10 (depends on 9)

The method of claim 9, wherein said target molecule or said phage display library is bound to or attached to a solid support.

### Claim 11 (depends on 9)

The method of claim 9, wherein said target molecule is BAFF, TfR or myostatin.