US 12,139,543 B2
Method of using an anti-epidermal growth factor receptor (EGFR) activatable antibody to detect the presence or absence of a cleaving agent and EGFR
Henry Bernard Lowman, El Granada, CA (US); Luc Roland Desnoyers, San Francisco, CA (US); and Shouchun Liu, Burlingame, CA (US)
Assigned to CytomX Therapeutics, Inc.
Filed by CytomX Therapeutics, Inc., South San Francisco, CA (US)
Filed on Nov. 22, 2021, as Appl. No. 17/532,193.
Application 17/532,193 is a division of application No. 16/712,422, filed on Dec. 12, 2019, granted, now 11,180,562.
Application 16/712,422 is a division of application No. 15/382,890, filed on Dec. 19, 2016, granted, now 10,533,053, issued on Jan. 14, 2020.
Application 15/382,890 is a division of application No. 14/528,180, filed on Oct. 30, 2014, granted, now 9,540,440, issued on Jan. 10, 2017.
Claims priority of provisional application 61/897,465, filed on Oct. 30, 2013.
Claims priority of provisional application 61/897,418, filed on Oct. 30, 2013.
Claims priority of provisional application 61/897,442, filed on Oct. 30, 2013.
Claims priority of provisional application 61/897,385, filed on Oct. 30, 2013.
Claims priority of provisional application 61/897,391, filed on Oct. 30, 2013.
Prior Publication US 2022/0289853 A1, Sep. 15, 2022
Int. Cl. C07K 16/28 (2006.01); A61K 39/395 (2006.01); A61K 47/68 (2017.01); G01N 33/564 (2006.01); G01N 33/574 (2006.01)
CPC C07K 16/2863 (2013.01) [A61K 39/39533 (2013.01); A61K 47/6817 (2017.08); A61K 47/6849 (2017.08); G01N 33/564 (2013.01); G01N 33/57492 (2013.01); C07K 2317/14 (2013.01); C07K 2317/24 (2013.01); C07K 2317/51 (2013.01); C07K 2317/515 (2013.01); C07K 2317/54 (2013.01); C07K 2317/55 (2013.01); C07K 2317/569 (2013.01); C07K 2317/76 (2013.01); C07K 2317/92 (2013.01); C07K 2319/50 (2013.01); G01N 2333/71 (2013.01); G01N 2800/205 (2013.01)] 25 Claims
 
1. A method of detecting presence or absence of a cleaving agent and Epidermal Growth Factor Receptor (EGFR) in a subject or a sample, the method comprising:
(a) contacting a subject or a sample with an anti-EGFR activatable antibody comprising
an antibody or an antigen-binding fragment thereof (AB) that specifically binds to EGFR, wherein the AB comprises
(I) a heavy chain comprising:
(i) heavy chain complementarity determining regions (HCDRs), wherein the HCDRs of the AB are the HCDRs of the heavy chain amino acid sequence of SEQ ID NO: 2, and
(ii) at least one of the following amino acid sequence features: (1) the heavy chain of the AB has a glutamine residue at the position corresponding to position 88 of SEQ ID NO: 2, (2) the heavy chain of the AB has an alanine residue at the position corresponding to position 299 of SEQ ID NO: 2, and (3) the heavy chain of the AB has a lysine residue at the position corresponding to position 216 of SEQ ID NO: 2;
(II) a light chain comprising:
(i) light chain complementarity determining regions (LCDRs), wherein the LCDRs are the LCDRs of the light chain amino acid sequence of SEQ ID NO: 32; and
(ii) a spacer sequence comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 14, 16, 18, 20, and 22,
wherein the light chain amino acid sequence is coupled to a masking moiety (MM) via a cleavable moiety (CM) to produce a masked light chain,
wherein the MM comprises the amino acid sequence of SEQ ID NO: 76, wherein the MM inhibits the binding of the AB of the anti-EGFR activatable antibody in an uncleaved state to EGFR,
wherein the CM is a polypeptide that functions as a substrate for a protease, and
wherein the anti-EGFR activatable antibody in the uncleaved state has a structural arrangement from N-terminus to C-terminus as follows: Spacer Sequence-MM-Linking Peptide 1 (LP1)-CM-Linking Peptide 2 (LP2)-AB or AB LP2 CM LP1 MM Spacer Sequence; and
(b) measuring a level of activated anti-EGFR activatable antibody in the subject or sample,
wherein a detectable level of activated anti-EGFR activatable antibody in the subject or sample indicates that the cleaving agent and EGFR are present in the subject or sample, and wherein no detectable level of activated anti-EGFR activatable antibody in the subject or sample indicates that the cleaving agent, EGFR or both the cleaving agent and EGFR are absent in the subject or sample.