1. An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody
Jens C. Krause, Damian C. Ekiert, Terrence M. Tumpey, Patricia B. Smith, Ian A. Wilson, and James E. Crowe, Jr.
Paige Byerly
Aman Kaur
Leah McElvain

2. Introduction Mab 2D1 IgG1/λ human monoclonal antibody (Mab)
Derived from B cell in 1918 influenza pandemic survivor
Antigenically similar to influenza A H1N1 virus
Binds to both the 1918 and 2009 influenza viruses
H1N1 Virus

3. Introduction Mab 2D1 Derived from human VH2-70 gene segment
o Undergone:
+ point mutations
+ 9-bp insertion in framework
3 of heavy chain
Mab del 2D1
9-bp insertion was removed
Reverted wild-type VH2-70 gene segment
Comparison of Mab 2D1 and Mab del 2D1
Antibody

4. Introduction Questions:
What is the genetic mechanism resulting in the Mab 2D1 insertion?
Does the insertion affect binding affinity and if it enhances antibody binding, why?
Does removing the insertion affect the functional activity of the antibody in vitro and in vivo?

5. Methods Animal studies. Sequence accession Numbers.
Expression and purification of wt 2D1 and del 2D1.
HAI Assays
HAI test using full length recombinant purified
Mabs were performed.

6. Results
Genetics mechanism of insertion: Somatic point mutation (Fig 2).
Hot Spot motifs resulting from mutation introduced that occurs at the site leading to duplication.
The difference in binding site was mediated only by the insertion.
When tested in vivo antiviral del2d1 was less effective than wt 2d1 at every dose tested.

7. FIG 2 Predicted genetic mechanism of the naturally occurring 2D1 somatic insertion/deletion. (A)
Nucleotide sequences surrounding the insertion/deletion site of MAb 2D1 and its predicted germ line
VH gene segment sequence, VH2-70. The adjacent nucleotide sequence to which the insertion/deletion
is related is in bold. Shaded boxes highlight the repeat nucleotide sequence present prior to insertion/
deletion. The nucleotides in the inserted sequence that likely were mutated before (in italics) or after
(underlined) the insertion/deletion are indicated. (B) For the insertion to occur, the misaligned repeat
sequence (in bold) created a loop and a misaligned “foot” that was extended subsequently. The nucleotides
that form the basis of the deletion in panel C are in red in panel B, part 5. (C) Similarly, a
misalignment in the antisense strand can lead to a deletion (according to de Wildt et al. [1]).

8. Results
In viro, the group of six mice receiving the highest dose level of the MAbs (del 2D1 and wt 2D1 at 200 ug) were protected from the 1918 influenza, as opposed to the control group, which did not receive anything and did not survive.
In contrast, in the group receiving the intermediate dose of the MAbs (20 ug of antibodies de2D1 and wt 2D), four out of six of the animals died from infection, while animals from the wt 2D1 treated group all survived.

9. Results
FIG 3 Therapeutic efficacy of 1918 HA-specific MAbs against disease caused
by the 1918 A(H1N1) virus in mice. Mice were inoculated on day 0 and treated
on day 1 with the indicated antibody and dose. In each group, six mice were
monitored every other day for weight (A) and survival (B).

10. Discussion Why is mutation so common in antibody DNA?
Insertions and deletions result from somatic hypermutation process:
Errors can occur from a double-strand break during DNA replication or
Errors can occur from double-stranded breaks during DNA transcription
and
Errors are both hot-spot and randomly directed

11. Discussion
How does the presence of insertions and deletions enhance antibody binding?
The example of MAb 2D1reveals new principles about the generation of antibodies:
The effect of insertion is indirect:
Insertion near one CDR loop affects/moves adjacent CDR loop
The effect of the insertion is subtractive:
CDR loop displaced from/frees up region of interaction
Important for antibody affinity maturation:
Removing reactions can be as important as adding ones

12. FIG 5 Combining site reconfiguration due to ITY insertion relieves potential steric clashes between the
epitope on HA (bottom left in grey and cyan) and antibody CDRs (top right). Superposition of del 2D1
Fab onto the wt 2D1-HA complex structure, with Fabs oriented approximately as in Fig. 4. In the germ
line configuration, the base of CDR H1 and the Trp52a side chain from CDR H2 would be in conflict
with an HA epitope loop. The ITY insertion and extended CDR H2 in wt 2D1 push Trp52a up and away
from the epitope, simultaneously displacing CDR H1 from its usual position, allowing wt 2D1 to
recognize HA in a manner that is otherwise unfavorable for the germ line Fab.

13. Discussion How does insertion move CDR chains?
Insertion is within a beta sheet—should disrupt, maybe even destabilize the protein
But…
Instead, insertions displace coding residues in CDR H2, move CDR H1 instead

14. …in conclusion,
Somatic insertions in the genes of antibodies may play a key role in maintaining the potency of these antibodies, as well as promoting their diversity. This may be an important tool in further studies of human pathogens and the body’s innate ability to defend itself against them.

15. Work Cited
Jens C. Krause, Damian C. Ekiert, Terrence M. Tumpey, Patricia B. Smith, Ian A. Wilson, and James E. Crowe, Jr An Insertion Mutation That
Distorts Antibody Binding Site Architecture Enhances Function of a
Human Antibody. mBio 2(1): 1-8.