1. It’s Nothing to Sneeze At – An Update in Allergy
Virginia Chapter ACP Annual Meeting
2013
Scott P. Commins
Division of Allergy and Clinical Immunology
Cetuximab:
Marketed under Erbitux
Chimeric monoclonal IgG1 antibody against the epidermal growth factor receptor (EGFR)
Used primarily in treatment of metastatic colorectal cancer and head and neck cancer
Discovered by ImClone, distributed by BMS, and costs ~$30K per 8 wks of tx
Disproportionate HSR in parts of the South

2. Disclosures We are grateful for generous support from
the Oakey Food Allergy Fund
These studies were carried out with generous support from the NIH-NIAID (K08 AI & R21 AI087985)
Disclosure: Up-to-Date card

3. How oncologists helped uncover a novel food allergy
I. Background
How oncologists helped uncover a novel food allergy

4. High incidence of hypersensitivity reactions to cetuximab
infusions in mid-Missouri:
Association with prior history of atopy
R. Owera, A. Gill, S. Haddadin, R. Khozouz and M. C. Perry
University of Missouri Columbia, Columbia, MO
Abstract, 2008 ASCO Annual Meeting Proceedings, Vol 26, No 15S, 2008:20747
UNC clinical trial data for 88 patients,
22% had grade 3 to 4 HSR
Data at that time from 400 patients in EPIC trial:
any infusion reaction = 4%, grade 3 to 4 = 0.5%
All reactions occurred with the first dose
Assessment Tools
Table 1. Common Toxicity Criteria (ver 3.0) of the National Cancer Institute
Grade 1 Transient flushing or rash; Drug fever <100.4°F
Grade 2 Rash; urticaria; dyspnea; Drug fever >100.4°F and/or asymptomatic bronchospasm
Grade 3 Symptomatic bronchospasm with or without urticaria; parenteral medication(s) indicated; allergy-related edema/anigiodema
Grade 4 Anaphylaxis
Grade 5 Death
Data from National Cancer Institute Cancer Therapy Evaluation Program.8

5. Regarding cetuximab HSR:
The story got stranger as O’Neil talked to more oncologists. He heard that a colleague in Nashville, Tennessee, was finding the same problem. But when O’Neil spoke to oncologists from other areas of the country, they didn’t know what he was talking about. A prominent colorectal oncologist in New York “thought we were lying or crazy,” O’Neil recalls.
From “A Mysterious Allergy Afflicts the South” by Sheila Read in Endeavors, 24:2, 2-3, 2008.

6. Measurement of IgE ab to Purified Allergens
Using Streptavidin Solid Phase
“Allergen” of interest (cetuximab)
+ Biotin
Streptavidin Solid Phase + Biotinylated “Allergen”
Solid Phase*
Then add serum; wash; add labeled anti-IgE; detect chemilum.
*Routine assay technique in parallel with Phadia standard curve gives results in IU/ml.

7. IgE Antibodies Binding to Cetuximab in Sera
from 76 Case Subjects and 462 Controls
Figure 1. IgE Antibodies Binding to Cetuximab in Serum Samples from 76 Case Subjects and 462 Control Subjects. Results are shown according to whether the treating physician reported a hypersensitivity reaction (HSR) to cetuximab or no HSR reaction. Results are also shown for pretreatment serum samples from control subjects and from subjects who had not received cetuximab. The horizontal lines indicate geometric mean values for the positive results. Values with multiplication signs indicate the number of negative values for each symbol.

8. Typical Fc region oligos: core fucosylated biantennary oligos;
Cetuximab *
Site not glycosylated
N-88
N-43
N-299
Qian J, Liu T,…Zhou Q.
Analytical Biochem, 2007.
Typical Fc region oligos: core fucosylated biantennary oligos;
no alpha-gal or NGNA
C’tux is an IgG1, human/mouse chimeric monoclonal ab against the EGFR and is produced in the SP2/0 mammalian (murine myeloma) cell line.
Variable domain is glycosylated in approx 20% of human IgGs.
21 distinct oligosaccharide structures in c’tux. These oligos differ mainly in degree of sialylation with N-glycolyl neuraminic acid and extent of galactosylation. Of the 21 oligos identified, approx 30% have one or more alpha-1,3 linked galactosyl residues as measured by peak area on TOF-MS spectra. Approx 12% have an NGNA residue and a very small amount of high mannose. Both alpha-gal and NGNA were found only in the Fab portion.
Unique among therapeutic mAbs, c’tux has 2 occupied N-linked glycosylation sites on the heavy chain: Asn88 & Asn299 (usually 297)

9. Specificity of the IgE Antibodies that Cross-React with Cetuximab
Table 3. Specificity of the IgE Antibodies That Cross-React with Cetuximab.
Chung CH et al. N Engl J Med 2008;358:

10. II. Defining a “new” food allergy

11. Specificity of the IgE Antibodies That Cross-React with Cetuximab
Chung CH et al. N Engl J Med 2008;358:
Specificity of the IgE Antibodies That Cross-React with Cetuximab
Table 3. Specificity of the IgE Antibodies That Cross-React with Cetuximab.
Contained w/n nejm publication is sera results from 6 patients with recurrent anaphylaxis not related to cetuximab.

12. Symptoms delayed 3-6 hours after eating mammalian meat
JACI, February 2009
24 patients
Virginia & Missouri
Symptoms delayed 3-6 hours after eating mammalian meat
Prick skin test often less than 4mm
Intradermal skin test positive

13. Skin Testing Results: Often <4mm SPT
Prick test
Intradermal
Prick test performed using lancette and intradermal testing with 25 gauge needle in the same patient on a single clinic visit.

14. Patient data, presentation, symptom delay, and laboratory evaluation
Age /Sex
Race
Symptoms
ANA = Anaphylaxis
AE = Angioedema
U = Urticaria
Time to Reaction (hours)
Alpha-Gal IgE Titer
(IU/mL)
Beef IgE Titer (IU/mL)
Total IgE Titer (IU/mL)
E001
44 / M W
ANA 6
 67.0
3.48
157
E008
80 / F
AE, U
 0.52
0.35
45.9
E018
26 / M 5
59.3
16.6
274
E022
74 / F 3
 9.03
8.90
66.6
E026
47 / M
 >100
32.9
1081
E030
56 / M U
4-6
 80.6
15.8 
709
E031
55 / F 4
 68.9
26.2
243
E036
66 / M
21.1 
19.2
262
E054
45 / M
2.42
244
E076
50 / M
 9.25
3.24
247
E115
58 / F
2-4
47.8
1622
E128
37 / M
26.8
5.65
886
E162
39 / F
11.1
1.51
30.5

15. CAP-RAST of Specific IgE Abs in Patients with anti-Gal IgE
Foods
Inhalants
r =0.81
r =0.98
Specific IgE Abs (IU/mL)
Limit of
detection
▼x1
♦x1
x3
x12
x11
x12
x1
x7
x1
x9
♦x4

16. Anaphylaxis/ urticaria subjects
Alpha-gal specific IgE is associated with anaphylaxis but not asthma (Figure adapted from Commins, Kelly…Platts-Mills, AJRCCM, 2012)
0.35
0.1 1 10
100
1000
= Reactions include anaphylaxis
Alpha-gal specific IgE (IU/mL)
We are exceedingly grateful to allergists and colleagues throughout the US and beyond for finding cases and sending samples. This is truly a team effort and we appreciate the support.
Limit of
detection
• x 8
• x 13
• x 58
• x 55
Number <0.35
Cat in home
(N = 96)
No cat in home
(N = 112)
Asthma
(N = 68)
Clinic controls
(N = 59)
Anaphylaxis/ urticaria subjects 16

17. 30%
10%
r = 0.60, p<0.001
Figure 2

18. IgE Ab to alpha-gal in kids (n=45)
Sex (% male)
69%
Mean age at presentation
(Range)
12 (4-17)
Geometric Mean of Total IgE (95% CI)
147 IU/mL ( IU/mL)
Symptoms at presentation
Anaphylaxis*
44%
Gastrointestinal/
Oral
64%
Urticaria
92%
Angioedema
31%
Average time to symptoms (Range)
4.68 hrs (10 mins to 24 hrs)
Tick exposure
100%
Redness and
Itching at site of
Tick bite
87%
Tick borne illness#
10%
Emergency Department (ED) visits
46%**
Medications administered in ED
Epinephrine (19%)
Antihistamines (35%)
Oral Steroids (19%)
Parenteral Steroids (17%)
IV Fluids (17%)
Hospital Admissions 8%

19. IgE Ab to alpha-gal in kids (n=45)

20. Delayed symptoms induced by oral exposure to mammalian proteins in patients with IgE ab to alpha-gal.
Despite high titer IgE antibodies that bind epitopes on a range of mammalian proteins including beef, pork and lamb:
No awareness of immediate response in the mouth (i.e. no lip swelling or tingling).
Anaphylaxis occurring after eating beef starts with skin itching 3-6 hours later.
Skin prick tests generally <4mm in diameter while intradermals often ≥8mm.

21. ‘Delayed’ food allergy?
Food allergy symptoms classically occur immediately (OAS), up to 2 hours after eating
No mention of ‘delayed’ symptoms that can be attributed to food in common allergy texts

22. III. Ticks. Really?

23. Geographical distribution of cetuximab hypersensitivity reactions
As reported by O’Neil et al, JCO 2007

24. Distribution of known cases of delayed anaphylaxis to mammalian meat
Dots = single cases
Smaller stars = 5 to 24 cases within a state
Larger stars = states with 25 or more cases

25. Geographical Incidence of Rocky Mountain Spotted Fever
Data from CDC website for 2009; accessed 2/2011

26. Original image: CDC; modified by D. N
Original image: CDC; modified by D.N. Gaines for Virginia Department of Health

27. Geographical range of Amblyomma americanum population
Data from CDC website; accessed 10/2010

28. Geographical range of Amblyomma americanum population
welcome to the party
Data from CDC website; accessed 9/2012

29. Bites from the larvae of Amblyomma or Dermacentor ticks and IgE ab to alpha-Gal.
Test
10/26/06
5/21/07
10/9/07
11/6/07*
Cetuximab
(alphaGal)
0.49
16.9
48.3
130
Total IgE
127
199
350
532
Multiple bites by seed ticks are not uncommon in the Southeast.
*Titers:Total 262, Beef 19.2 Pork 10.1,Cat 17.5,Dog 19.8, IU/ml. Chicken, Turkey, Roach, Dust Mite and Ragweed all <0.35 IU/ml.
Photograph: August 23, 2007.

30. Time course of IgE antibodies to alpha-gal and total IgE
following episodes of multiple tick bites (red arrows) *
Subject #1
Fig 1, Panel 1

31. Time course of IgE antibodies to alpha-gal and total IgE
following episodes of multiple tick bites (red arrows)
Subject #2
Fig 1, Panel 2

32. Time course of IgE antibodies to alpha-gal and total IgE
following episodes of multiple tick bites (red arrows) *
Subject #3
Fig 1, Panel 3

33. Total IgE versus IgE to Alpha-Gal
Report tick or chigger bite(s)
No history obtained for tick bite(s)
Specifically deny tick bite(s)
1000
IgE to Alpha-Gal (IU / mL)
R= 0.78
P <0.001
100 10 1
0.1 1 10
100
1000
10000
Total IgE (IU / mL)

34. R amblyommii?
Overall infection prevalences were 4.7% for E. chaffeensis (range, 0Ð27%), 3.5% for E. ewingii (range, 0Ð18.6%), 2.5% for B. lonestari (range, 0Ð12.2%), and 41.2% for R. amblyommii (range, 0Ð84.0%).

35. IV. Delayed food allergy

37. -FC02 - sIgE to alpha-gal = 22.4 IU/mL; Total IgE = 109
-4:22 after consuming meat, symptoms began as itching & hive on hand; second hive appeared on arm
-Given AH and observed

38. -FC02 - sIgE to alpha-gal = 17.6 IU/mL; Total IgE = 184
-Whole blood was collected, fixed & stained
-3:54 after consuming meat, symptoms began as itching, flushing and progressed: ultimately requiring epinephrine

39. -Subject #FC-07: sIgE to alpha-gal = 9.3 IU/mL; Total IgE = 204
-Whole blood was collected, fixed & stained
-6 hours after consuming 56g of pork prosciutto, subject released with (disappointing) mild itch and single hive

40. Subject calls from car 30 minutes after being released to report progression of itching and “warmth to skin”
Initial picture upon arriving home (approximately 7.5 hours after eating pork prosciutto)

41. Approximately 8.25 hours after eating prosciutto
Samantha let’s the reaction proceed = they are not afraid. Others are b/c of ana

42. Mammalian meat challenge
Subject #FC-08: sIgE to alpha-gal = 30.3 IU/mL ; total IgE = 146
Before
3hrs 45min after eating meat

43. Diffuse urticaria appeared at 4hrs 15min after eating meat
Subject #FC-08: Mammalian meat challenge
Diffuse urticaria appeared at
4hrs 15min after eating meat
(left arm shown here)

44. Mammalian meat challenge
Subject #FC-10: sIgE to alpha-gal = 29.1 IU/mL ; total IgE = 201
4:25 in
4hrs 25min after eating mammalian meat: pruritus followed by urticaria on right flank

45. Summary of Mammalian Meat Challenge
Results in Subjects with IgE to alpha-gal
Urticaria
Time to symptoms (hrs:min)
Treatment required
Tryptase elevation
Allergics (n=10)
7*/10
3:50 – 6:00
7/10
1**/9
*Two subjects received epinephrine (i.e., these reactions are probably better defined as delayed anaphylaxis to mammalian meat)
**Tryptase:
-Subject FC-08: 4.2ng/mL (baseline) → 18.2ng/mL (4 hrs) → 20.1 ng/mL (5 hrs)
-Subject FC-10: 4.9 (baseline) → 6.2 (4 hrs) → 9.9 (5 hrs) → 10.7 (6 hrs)
-No tryptase measurements were performed in subject FC-06

46. Gating Strategy Gating Strategy Basophils % of max CD 123 Negative for
HLA-DR/lineage 1 FITC
CD63 APC
CD123 PerCP-Cy5.5
Basophils
Negative for
Lineage Cocktail 1 (CD3, CD14, CD16, CD19,CD20, and CD56)
HLA-DR
Positive for
CD123 (IL-3Ra)
FcεRI (IgE receptor)
IgE
CD 123
% of max

47. -APC -APC FACS Detection of CD63, a marker of Basophil Activation CD63
PerCP-Cy5.5-linked anti-CD123 ab
APC-linked
anti-CD63 ab
-APC
CD63
IL-3 Rα
-APC
Histamine
Shift to discuss flow
nuclear membrane

48. In vivo Basophil Activation during Mammalian Meat Challenge
1 hr
0.72%
2 hrs
0.54%
3 hrs
0.87%
CD63
CD123
In vivo Basophil Activation during Mammalian Meat Challenge
#FC-02
4 hrs %
5 hrs %
6 hrs %

49. In vivo Basophil Activation during Mammalian Meat Challenge
Baseline
1.96%
2 hrs
3.08%
4 hrs
1.8%
5 hrs
8.74%
-FC07 - sIgE to alpha-gal = 9.3 IU/mL; Total IgE = 204
-Whole blood was collected, fixed & stained
CD63
IgE

50. Summary of Mammalian Meat Challenge
Results in Subjects with or without IgE to alpha-gal
Delayed anaphylaxis
Urticaria
Time to symptoms (hrs:min)
Treatment required
Allergics
2/10
7/10
3:50 – 6:00
Controls
0/10
n/a
Delayed anaphylaxis
Urticaria
Time to symptoms (hrs:min)
Treatment required
Basophil activation
Allergics
2/10
6/10
3:50 – 6:00
7/10
8/10

51. Why the delay?
The answer could be fat. Recall that fat is absorbed differently from protein or carbohydrates in a process that takes 3-4 hrs to enter the blood stream

52. The delivery of lipid to the circulation takes time (about 4 hours)
VLDL
IDL
LDL
HDL
Further conversion
=Alpha-gal moiety,
as a glycolipid
Thoracic duct blood stream

53. Why the delay?
The answer could be long-chain triglycerides associated with chylomicrons. Studies suggest LCT enhance dietary antigen absorption into the circulation via chylomicron formation.
The answer could be the nature of a carbohydrate-based food allergy.

54. Conclusions
The report of delayed reactions occurring 3-6 hours after eating mammalian meat has been confirmed on multiple occasions and at two medical centers
In subjects with IgE to alpha-gal, basophil activation in vivo coincides with the appearance of clinical symptoms
Lack of basophil activation before 4 hours suggests that there is delayed appearance of the antigen into the bloodstream

55. Contributors Uva Hayley James Barrett Lewis Susan Mozzicato
Lisa Workman
Alicia Clark
Judith Woodfolk
Walter Oliveira
Whitney Stevens
Shama Satinover
Libby Kelly
Peter Heymann
Amy Stallings
Julia Wisniewski
Josh Kennedy
Anubha Tripathi
Jonathon Posthumus
Barrett Lewis
Michael Land
Joey Lane
Heather Gütekünst
Jane Purser
Gary Rakes
Madeline Dillon
Tina Hatley
Luis Matos
Saju Eapen
Dane McBride
Joseph Belleau
Harold Moessner
Marty Fiser
Eddie Shields