Internalizing moieties
The present disclosure provides antibodies and antigen-binding fragments capable of penetrating cells via an ENT transporter, such as humanized antibodies. The disclosure also provides conjugates comprising the antibodies or antigen-binding fragments and a heterologous agent. The disclosure further provides methods for making and using the antibodies, antigen-binding fragments and conjugates, including humanized antibodies.
1. An antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a light chain variable (VL) domain and a heavy chain variable (VH) domain; wherein the VH domain comprises:
a VH CDR1 having the amino acid sequence of SEQ ID NO: 32;
a VH CDR2 having the amino acid sequence of SEQ ID NO: 49; and
a VH CDR3 having the amino acid sequence of SEQ ID NO: 34,
which CDRs are according to the Kabat system;
and the VL comprises:
a VL CDR1 having the amino acid sequence of SEQ ID NO: 35 or 50;
a VL CDR2 having the amino acid sequence of SEQ ID NO: 51; and
a VL CDR3 having the amino acid sequence of SEQ ID NO: 37,
which CDRs are according to the Kabat system;
wherein the antibody or antigen-binding fragment binds DNA.
2. The antibody or antigen binding fragment of claim 1 , wherein the VH domain and VL domain are humanized.
3. The antibody or antigen-binding fragment of claim 1 , wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 40.
4. The antibody or antigen-binding fragment of claim 1 , wherein the VL comprises the amino acid sequence set forth in SEQ ID NO: 8.
5. The antibody or antigen-binding fragment of claim 1 , wherein the V H domain comprises the amino acid sequence set forth in SEQ ID NO: 39.
6. The antibody or antigen-binding fragment of claim 1 , wherein the V H domain comprises the amino acid sequence set forth in SEQ ID NO: 10.
7. The antibody or antigen-binding fragment of claim 1 , wherein the VH comprises the amino acid sequence set forth in SEQ ID NOs: 10, 38 or 39.
8. An antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment comprises a light chain variable (V L ) domain and a heavy chain variable (V H ) domain; wherein the V L domain comprises the amino acid sequence set forth in SEQ ID NO: 8; and the VH domain comprises the amino acid sequence set forth in SEQ ID NO: 10, wherein the antibody or antigen-binding fragment binds DNA and penetrates cells.
9. The antibody or antigen-binding fragment of claim 1 , wherein the V H domain comprises one or more of the following amino acid alterations: V5Q, E6Q, L11V, V12I, K13Q, R18L, K19R, V37I, E42G, A49S, T63S, A75S, F80Y, T84N, S88A, M93V, T111L or L112V, as compared with and numbered with respect to the amino acid sequence of SEQ ID NO: 9.
10. The antibody or antigen-binding fragment of claim 1 wherein the V L domain comprises one or more of the following amino acid alterations: V3Q, L4M, A9S, A12S, V13A, L15V, Q17D, A19V, S22T, M37L, H38A, G45E, Q46K, P47A, E59Q, A64S, H76T, N78T, H80S, P81S, V82L, E83Q, E84P, A87V, A87F, or G104A, as compared with and numbered with respect to the amino acid sequence of SEQ ID NO: 7.
11. The antibody or antigen-binding fragment of claim 1 , which is an scFv.
12. The antibody or antigen-binding fragment of claim 1 , which is a Fab′.
13. The antibody or antigen-binding fragment of claim 1 , which is a full length antibody comprising a heavy chain constant domain and a light chain constant domain.
14. The antibody or antigen-binding fragment of claim 1 , wherein the antibody or antigen-binding fragment is conjugated to a heterologous agent.
15. A composition comprising the antibody or antigen-binding fragment of claim 1 formulated in a physiologically acceptable carrier.
16. The composition of claim 15 , wherein the composition is pyrogen free.